Metabolic Deficits in the Retina of a Familial Dysautonomia Mouse Model.

Neurodegenerative retinal diseases such as glaucoma, diabetic retinopathy, Leber's hereditary optic neuropathy (LHON), and dominant optic atrophy (DOA) are marked by progressive death of retinal ganglion cells (RGC). This decline is promoted by structural and functional mitochondrial deficits, including electron transport chain (ETC) impairments, increased oxidative stress, and reduced energy (ATP) production. These cellular mechanisms associated with progressive optic nerve atrophy have been similarly observed in familial dysautonomia (FD) patients, who experience gradual loss of visual acuity due to the degeneration of RGCs, which is thought to be caused by a breakdown of mitochondrial structures, and a disruption in ETC function. Retinal metabolism plays a crucial role in meeting the elevated energetic demands of this tissue, and recent characterizations of FD patients' serum and stool metabolomes have indicated alterations in central metabolic processes and potential systemic deficits of taurine, a small molecule essential for retina and overall eye health. The present study sought to elucidate metabolic alterations that contribute to the progressive degeneration of RGCs observed in FD. Additionally, a critical subpopulation of retinal interneurons, the dopaminergic amacrine cells, mediate the integration and modulation of visual information in a time-dependent manner to RGCs. As these cells have been associated with RGC loss in the neurodegenerative disease Parkinson's, which shares hallmarks with FD, a targeted analysis of the dopaminergic amacrine cells and their product, dopamine, was also undertaken. One dimensional (1D) proton (1H) nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and retinal histology methods were employed to characterize retinae from the retina-specific Elp1 conditional knockout (CKO) FD mouse model (Pax6-Cre; Elp1LoxP/LoxP). Metabolite alterations correlated temporally with progressive RGC degeneration and were associated with reduced mitochondrial function, alterations in ATP production through the Cahill and mini-Krebs cycles, and phospholipid metabolism. Dopaminergic amacrine cell populations were reduced at timepoints P30-P90, and dopamine levels were 25-35% lower in CKO retinae compared to control retinae at P60. Overall, this study has expanded upon our current understanding of retina pathology in FD. This knowledge may apply to other retinal diseases that share hallmark features with FD and may help guide new avenues for novel non-invasive therapeutics to mitigate the progressive optic neuropathy in FD.

Primary Human M2 Macrophage Subtypes Are Distinguishable by Aqueous Metabolite Profiles.

The complexity of macrophage (MΦ) plasticity and polarization states, which include classically activated pro-inflammatory (M1) and alternatively activated anti-inflammatory (M2) MΦ phenotypes, is becoming increasingly appreciated. Within the M2 MΦ polarization state, M2a, M2b, M2c, and M2d MΦ subcategories have been defined based on their expression of specific cell surface receptors, secreted cytokines, and specialized immune effector functions. The importance of immunometabolic networks in mediating the function and regulation of MΦ immune responses is also being increasingly recognized, although the exact mechanisms and extent of metabolic modulation of MΦ subtype phenotypes and functions remain incompletely understood. In this study, proton (1H) nuclear magnetic resonance (NMR) metabolomics was employed to determine the polar metabolomes of M2 MΦ subtypes and to investigate the relationship between aqueous metabolite profiles and M2 MΦ functional phenotypes. Results from this study demonstrate that M2a MΦs are most distinct from M2b, M2c, and M2d MΦ subtypes, and that M2b MΦs display several metabolic traits associated with an M1-like MΦ phenotype. The significance of metabolome differences for metabolites implicated in glycolysis, the tricarboxylic acid (TCA) cycle, phospholipid metabolism, and creatine-phosphocreatine cycling is discussed. Altogether, this study provides biochemical insights into the role of metabolism in mediating the specialized effector functions of distinct M2 MΦ subtypes and supports the concept of a continuum of macrophage activation states rather than two well-separated and functionally distinct M1/M2 MΦ classes, as originally proposed within a classical M1/M2 MΦ framework.

Metabolomic profiling of human bladder tissue extracts.

INTRODUCTION: Bladder cancer is a common malignancy affecting the urinary tract and effective biomarkers and for which monitoring therapeutic interventions have yet to be identified. OBJECTIVES: Major aim of this work was to perform metabolomic profiling of human bladder cancer and adjacent normal tissue and to evaluate cancer biomarkers. METHODS: This study utilized nuclear magnetic resonance (NMR) and high-resolution nanoparticle-based laser desorption/ionization mass spectrometry (LDI-MS) methods to investigate polar metabolite profiles in tissue samples from 99 bladder cancer patients. RESULTS: Through NMR spectroscopy, six tissue metabolites were identified and quantified as potential indicators of bladder cancer, while LDI-MS allowed detection of 34 compounds which distinguished cancer tissue samples from adjacent normal tissue. Thirteen characteristic tissue metabolites were also found to differentiate bladder cancer tumor grades and thirteen metabolites were correlated with tumor stages. Receiver-operating characteristics analysis showed high predictive power for all three types of metabolomics data, with area under the curve (AUC) values greater than 0.853. CONCLUSION: To date, this is the first study in which bladder human normal tissues adjacent to cancerous tissues are analyzed using both NMR and MS method. These findings suggest that the metabolite markers identified in this study may be useful for the detection and monitoring of bladder cancer stages and grades.

Targeted and untargeted urinary metabolic profiling of bladder cancer.

Bladder cancer (BC) is frequent cancer affecting the urinary tract and is one of the most prevalent malignancies worldwide. No biomarkers that can be used for effective monitoring of therapeutic interventions for this cancer have been identified to date. This study investigated polar metabolite profiles in urine samples from 100 BC patients and 100 normal controls (NCs) using nuclear magnetic resonance (NMR) and two methods of high-resolution nanoparticle-based laser desorption/ionization mass spectrometry (LDI-MS). Five urine metabolites were identified and quantified using NMR spectroscopy to be potential indicators of bladder cancer. Twenty-five LDI-MS-detected compounds, predominantly peptides and lipids, distinguished urine samples from BC and NCs individuals. Level changes of three characteristic urine metabolites enabled BC tumor grades to be distinguished, and ten metabolites were reported to correlate with tumor stages. Receiver-Operating Characteristics analysis showed high predictive power for all three types of metabolomics data, with the area under the curve (AUC) values greater than 0.87. These findings suggest that metabolite markers identified in this study may be useful for the non-invasive detection and monitoring of bladder cancer stages and grades.

A Comprehensive NMR Analysis of Serum and Fecal Metabolites in Familial Dysautonomia Patients Reveals Significant Metabolic Perturbations.

Central metabolism has a profound impact on the clinical phenotypes and penetrance of neurological diseases such as Alzheimer's (AD) and Parkinson's (PD) diseases, Amyotrophic Lateral Sclerosis (ALS) and Autism Spectrum Disorder (ASD). In contrast to the multifactorial origin of these neurological diseases, neurodevelopmental impairment and neurodegeneration in Familial Dysautonomia (FD) results from a single point mutation in the ELP1 gene. FD patients represent a well-defined population who can help us better understand the cellular networks underlying neurodegeneration, and how disease traits are affected by metabolic dysfunction, which in turn may contribute to dysregulation of the gut-brain axis of FD. Here, 1H NMR spectroscopy was employed to characterize the serum and fecal metabolomes of FD patients, and to assess similarities and differences in the polar metabolite profiles between FD patients and healthy relative controls. Findings from this work revealed noteworthy metabolic alterations reflected in energy (ATP) production, mitochondrial function, amino acid and nucleotide catabolism, neurosignaling molecules, and gut-microbial metabolism. These results provide further evidence for a close interconnection between metabolism, neurodegeneration, and gut microbiome dysbiosis in FD, and create an opportunity to explore whether metabolic interventions targeting the gut-brain-metabolism axis of FD could be used to redress or slow down the progressive neurodegeneration observed in FD patients.

Gut microbiome dysbiosis drives metabolic dysfunction in Familial dysautonomia.

Familial dysautonomia (FD) is a rare genetic neurologic disorder caused by impaired neuronal development and progressive degeneration of both the peripheral and central nervous systems. FD is monogenic, with >99.4% of patients sharing an identical point mutation in the elongator acetyltransferase complex subunit 1 (ELP1) gene, providing a relatively simple genetic background in which to identify modifiable factors that influence pathology. Gastrointestinal symptoms and metabolic deficits are common among FD patients, which supports the hypothesis that the gut microbiome and metabolome are altered and dysfunctional compared to healthy individuals. Here we show significant differences in gut microbiome composition (16 S rRNA gene sequencing of stool samples) and NMR-based stool and serum metabolomes between a cohort of FD patients (~14% of patients worldwide) and their cohabitating, healthy relatives. We show that key observations in human subjects are recapitulated in a neuron-specific Elp1-deficient mouse model, and that cohousing mutant and littermate control mice ameliorates gut microbiome dysbiosis, improves deficits in gut transit, and reduces disease severity. Our results provide evidence that neurologic deficits in FD alter the structure and function of the gut microbiome, which shifts overall host metabolism to perpetuate further neurodegeneration.

Ag(III)···Ag(III) Argentophilic Interaction in a Cofacial Corrole Dyad.

Metallophilic interactions between closed-shell metal centers are exemplified by d10 ions, with Au(I) aurophilic interactions as the archetype. Such an interaction extends to d8 species, and examples involving Au(III) are prevalent. Conversely, Ag(III) argentophilic interactions are uncommon. Here, we identify argentophilic interactions in silver corroles, which are authentic Ag(III) species. The crystal structure of a monomeric silver corrole is a dimer in the solid state, and the macrocycle exhibits an atypical domed conformation. In order to evaluate whether this represents an authentic metallophilic interaction or a crystal-packing artifact, the analogous cofacial or "pacman" corrole was prepared. The conformation of the monomer was recapitulated in the silver pacman corrole, exhibiting a short 3.67 Å distance between metal centers and a significant compression of the xanthene backbone. Theoretical calculations support the presence of a rare Ag(III)···Ag(III) argentophilic interaction in the pacman complex.

Distinct Metabolic States Are Observed in Hypoglycemia Induced in Mice by Ricin Toxin or by Fasting.

Hypoglycemia may be induced by a variety of physiologic and pathologic stimuli and can result in life-threatening consequences if untreated. However, hypoglycemia may also play a role in the purported health benefits of intermittent fasting and caloric restriction. Previously, we demonstrated that systemic administration of ricin toxin induced fatal hypoglycemia in mice. Here, we examine the metabolic landscape of the hypoglycemic state induced in the liver of mice by two different stimuli: systemic ricin administration and fasting. Each stimulus produced the same decrease in blood glucose and weight loss. The polar metabolome was studied using 1H NMR, quantifying 59 specific metabolites, and untargeted LC-MS on approximately 5000 features. Results were analyzed by multivariate analyses, using both principal component analysis (PCA) and partial least squares-discriminant analysis (PLS-DA), to identify global metabolic patterns, and by univariate analyses (ANOVA) to assess individual metabolites. The results demonstrated that while there were some similarities in the responses to the two stimuli including decreased glucose, ADP, and glutathione, they elicited distinct metabolic states. The metabolite showing the greatest difference was O-phosphocholine, elevated in ricin-treated animals and known to be affected by the pro-inflammatory cytokine TNF-α. Another difference was the alternative fuel source utilized, with fasting-induced hypoglycemia primarily ketotic, while the response to ricin-induced hypoglycemia involves protein and amino acid catabolism.

Metabolomic and elemental profiling of blood serum in bladder cancer.

Bladder cancer (BC) is one of the most frequently diagnosed types of urinary cancer. Despite advances in treatment methods, no specific biomarkers are currently in use. Targeted and untargeted profiling of metabolites and elements of human blood serum from 100 BC patients and the same number of normal controls (NCs), with external validation, was attempted using three analytical methods, i.e., nuclear magnetic resonance, gold and silver-109 nanoparticle-based laser desorption/ionization mass spectrometry (LDI-MS), and inductively coupled plasma optical emission spectrometry (ICP-OES). All results were subjected to multivariate statistical analysis. Four potential serum biomarkers of BC, namely, isobutyrate, pyroglutamate, choline, and acetate, were quantified with proton nuclear magnetic resonance, which had excellent predictive ability as judged by the area under the curve (AUC) value of 0.999. Two elements, Li and Fe, were also found to distinguish between cancer and control samples, as judged from ICP-OES data and AUC of 0.807 (in validation set). Twenty-five putatively identified compounds, mostly related to glycans and lipids, differentiated BC from NCs, as detected using LDI-MS. Five serum metabolites were found to discriminate between tumor grades and nine metabolites between tumor stages. The results from three different analytical platforms demonstrate that the identified distinct serum metabolites and metal elements have potential to be used for noninvasive detection, staging, and grading of BC.

Isolation and Characterization of Lignocellulose-Degrading Geobacillus thermoleovorans from Yellowstone National Park.

The microbial degradation of lignocellulose in natural ecosystems presents numerous biotechnological opportunities, including biofuel production from agricultural waste and feedstock biomass. To explore the degradation potential of specific thermophiles, we have identified and characterized extremophilic microorganisms isolated from hot springs environments that are capable of biodegrading lignin and cellulose substrates under thermoalkaline conditions, using a combination of culturing, genomics, and metabolomics techniques. Organisms that can use lignin and cellulose as a sole carbon source at 60 to 75°C were isolated from sediment slurry of thermoalkaline hot springs (71 to 81°C and pH 8 to 9) of Yellowstone National Park. Full-length 16S rRNA gene sequencing indicated that these isolates were closely related to Geobacillus thermoleovorans. Interestingly, most of these isolates demonstrated biofilm formation on lignin, a phenotype that is correlated with increased bioconversion. Assessment of metabolite level changes in two Geobacillus isolates from two representative springs were undertaken to characterize the metabolic responses associated with growth on glucose versus lignin carbon source as a function of pH and temperature. Overall, results from this study support that thermoalkaline springs harbor G. thermoleovorans microorganisms with lignocellulosic biomass degradation capabilities and potential downstream biotechnological applications. IMPORTANCE Since lignocellulosic biomass represents a major agro-industrial waste and renewable resource, its potential to replace nonrenewable petroleum-based products for energy production is considerable. Microbial ligninolytic and cellulolytic enzymes are of high interest in biorefineries for the valorization of lignocellulosic biomass, as they can withstand the extreme conditions (e.g., high temperature and high pH) required for processing. Of great interest is the ligninolytic potential of specific Geobacillus thermoleovorans isolates to function at a broad range of pH and temperatures, since lignin is the bottleneck in the bioprocessing of lignocellulose. In this study, results obtained from G. thermoleovorans isolates originating from YNP springs are significant because very few microorganisms from alkaline thermal environments have been discovered to have lignin- and cellulose-biodegrading capabilities, and this work opens new avenues for the biotechnological valorization of lignocellulosic biomass at an industrial scale.

Metabolomic and elemental profiling of blood serum in bladder cancer / 药物分析学报

Bladder cancer(BC)is one of the most frequently diagnosed types of urinary cancer.Despite advances in treatment methods,no specific biomarkers are currently in use.Targeted and untargeted profiling of metabolites and elements of human blood serum from 100 BC patients and the same number of normal controls(NCs),with external validation,was attempted using three analytical methods,i.e.,nuclear magnetic resonance,gold and silver-109 nanoparticle-based laser desorption/ionization mass spec-trometry(LDI-MS),and inductively coupled plasma optical emission spectrometry(ICP-OES).All results were subjected to multivariate statistical analysis.Four potential serum biomarkers of BC,namely,iso-butyrate,pyroglutamate,choline,and acetate,were quantified with proton nuclear magnetic resonance,which had excellent predictive ability as judged by the area under the curve(AUC)value of 0.999.Two elements,Li and Fe,were also found to distinguish between cancer and control samples,as judged from ICP-OES data and AUC of 0.807(in validation set).Twenty-five putatively identified compounds,mostly related to glycans and lipids,differentiated BC from NCs,as detected using LDI-MS.Five serum metab-olites were found to discriminate between tumor grades and nine metabolites between tumor stages.The results from three different analytical platforms demonstrate that the identified distinct serum metabolites and metal elements have potential to be used for noninvasive detection,staging,and grading of BC.

1H NMR based metabolic profiling distinguishes the differential impact of capture techniques on wild bighorn sheep.

Environmental metabolomics has the potential to facilitate the establishment of a new suite of tools for assessing the physiological status of important wildlife species. A first step in developing such tools is to evaluate the impacts of various capture techniques on metabolic profiles as capture is necessary to obtain the biological samples required for assays. This study employed 1H nuclear magnetic resonance (NMR)-based metabolite profiling of 562 blood serum samples from wild bighorn sheep to identify characteristic molecular serum makers of three capture techniques (dart, dropnet, and helicopter-based captures) to inform future sampling protocols for metabolomics studies, and to provide insights into the physiological impacts of capture. We found that different capture techniques induce distinct changes in amino acid serum profiles, the urea cycle, and glycolysis, and attribute the differences in metabolic patterns to differences in physical activity and stress caused by the different capture methods. These results suggest that when designing experiments involving the capture of wild animals, it may be prudent to employ a single capture technique to reduce confounding factors. Our results also supports administration of tranquilizers as soon as animals are restrained to mitigate short-term physiological and metabolic responses when using pursuit and physical restraint capture techniques.

Metabolomic and elemental profiling of human tissue in kidney cancer.

INTRODUCTION: Kidney cancer is one of the most frequently diagnosed and the most lethal urinary cancer. Despite advances in treatment, no specific biomarker is currently in use to guide therapeutic interventions. OBJECTIVES: Major aim of this work was to perform metabolomic and elemental profiling of human kidney cancer and normal tissue and to evaluate cancer biomarkers. METHODS: Metabolic and elemental profiling of tumor and adjacent normal human kidney tissue from 50 patients with kidney cancer was undertaken using three different analytical methods. RESULTS: Five potential tissue biomarkers of kidney cancer were identified and quantified using with high-resolution nuclear magnetic resonance spectroscopy. The contents of selected chemical elements in tissues was analyzed using inductively coupled plasma optical emission spectrometry. Eleven mass spectral features differentiating between kidney cancer and normal tissues were detected using silver-109 nanoparticle enhanced steel target laser desorption/ionization mass spectrometry. CONCLUSIONS: Our results, derived from the combination of ICP-OES, LDI MS and 1H NMR methods, suggest that tissue biomarkers identified herein appeared to have great potential for use in clinical prognosis and/or diagnosis of kidney cancer.

Nuclear magnetic resonance and surface-assisted laser desorption/ionization mass spectrometry-based metabolome profiling of urine samples from kidney cancer patients.

Kidney cancer is one of the most frequently diagnosed cancers of the urinary tract in the world. Despite significant advances in kidney cancer treatment, no urine specific biomarker is currently used to guide therapeutic interventions. In an effort to address this knowledge gap, metabolic profiling of urine samples from 50 patients with kidney cancer and 50 healthy volunteers was undertaken using high-resolution proton nuclear magnetic resonance spectroscopy (1H NMR) and silver-109 nanoparticle enhanced steel target laser desorption/ionization mass spectrometry (109AgNPET LDI MS). Twelve potential urine biomarkers of kidney cancer were identified and quantified using one-dimensional (1D) 1H NMR metabolomics. Seven mass spectral features which differed significantly in abundance (p < 0.05) between kidney cancer patients and healthy volunteers were also detected using 109AgNPET-based laser desorption/ionization mass spectrometry (LDI MS). This work provides a framework to expand biomarker discovery that could be used as useful diagnostic or prognostic of kidney cancer progression.

Pseudomonas aeruginosa Planktonic- and Biofilm-Conditioned Media Elicit Discrete Metabolic Responses in Human Macrophages.

Macrophages (MΦs) are prevalent innate immune cells, present throughout human bodily tissues where they orchestrate innate and adaptive immune responses to maintain cellular homeostasis. MΦs have the capacity to display a wide array of functional phenotypes due to different microenvironmental cues, particularly soluble bacterial secretory products. Recent evidence has emerged demonstrating that metabolism supports MΦ function and plasticity, in addition to energy and biomolecular precursor production. In this study, 1D 1H-NMR-based metabolomics was used to identify the metabolic pathways that are differentially altered following primary human monocyte-derived MΦ exposure to P. aeruginosa planktonic- and biofilm-conditioned media (PCM and BCM). Metabolic profiling of PCM- and BCM-exposed MΦs indicated a significant increase in glycolytic metabolism, purine biosynthesis, and inositol phosphate metabolism. In addition, these metabolic patterns suggested that BCM-exposed MΦs exhibit a hyperinflammatory metabolic profile with reduced glycerol metabolism and elevated catabolism of lactate and amino acids, relative to PCM-exposed MΦs. Altogether, our study reveals novel findings concerning the metabolic modulation of human MΦs after exposure to secretory microbial products and contributes additional knowledge to the field of immunometabolism in MΦs.

Metabolic Responses to Arsenite Exposure Regulated through Histidine Kinases PhoR and AioS in Agrobacterium tumefaciens 5A.

Arsenite (AsIII) oxidation is a microbially-catalyzed transformation that directly impacts arsenic toxicity, bioaccumulation, and bioavailability in environmental systems. The genes for AsIII oxidation (aio) encode a periplasmic AsIII sensor AioX, transmembrane histidine kinase AioS, and cognate regulatory partner AioR, which control expression of the AsIII oxidase AioBA. The aio genes are under ultimate control of the phosphate stress response via histidine kinase PhoR. To better understand the cell-wide impacts exerted by these key histidine kinases, we employed 1H nuclear magnetic resonance (1H NMR) and liquid chromatography-coupled mass spectrometry (LC-MS) metabolomics to characterize the metabolic profiles of ΔphoR and ΔaioS mutants of Agrobacterium tumefaciens 5A during AsIII oxidation. The data reveals a smaller group of metabolites impacted by the ΔaioS mutation, including hypoxanthine and various maltose derivatives, while a larger impact is observed for the ΔphoR mutation, influencing betaine, glutamate, and different sugars. The metabolomics data were integrated with previously published transcriptomics analyses to detail pathways perturbed during AsIII oxidation and those modulated by PhoR and/or AioS. The results highlight considerable disruptions in central carbon metabolism in the ΔphoR mutant. These data provide a detailed map of the metabolic impacts of AsIII, PhoR, and/or AioS, and inform current paradigms concerning arsenic-microbe interactions and nutrient cycling in contaminated environments.

Nuclear magnetic resonance and surface-assisted laser desorption/ionization mass spectrometry-based serum metabolomics of kidney cancer.

Kidney cancer is one of the most frequently diagnosed and the most lethal urinary cancer. Despite all the efforts made, no serum-specific biomarker is currently used in the clinical management of patients with this tumor. In this study, comprehensive high-resolution proton nuclear magnetic resonance spectroscopy (1H NMR) and silver-109 nanoparticle-enhanced steel target laser desorption/ionization mass spectrometry (109AgNPET LDI MS) approaches were conducted, in conjunction with multivariate data analysis, to discriminate the global serum metabolic profiles of kidney cancer (n = 50) and healthy volunteers (n = 49). Eight potential biomarkers have been identified using 1H NMR metabolomics and nine mass spectral features which differed significantly (p < 0.05) between kidney cancer patients and healthy volunteers, as observed by LDI MS. A partial least squares discriminant analysis (OPLS-DA) model generated from metabolic profiles obtained by both analytical approaches could robustly discriminate normal from cancerous samples (Q2 > 0.7), area under the receiver operative characteristic curve (ROC) AUC > 0.96. Compared with healthy human serum, kidney cancer serum had higher levels of glucose and lower levels of choline, glycerol, glycine, lactate, leucine, myo-inositol, and 1-methylhistidine. Analysis of differences between these metabolite levels in patients with different types and grades of kidney cancer was undertaken. Our results, derived from the combination of LDI MS and 1H NMR methods, suggest that serum biomarkers identified herein appeared to have great potential for use in clinical prognosis and/or diagnosis of kidney cancer. Graphical abstract.

Metabolic Implications of Using BioOrthogonal Non-Canonical Amino Acid Tagging (BONCAT) for Tracking Protein Synthesis.

BioOrthogonal Non-Canonical Amino acid Tagging (BONCAT) is a powerful tool for tracking protein synthesis on the level of single cells within communities and whole organisms. A basic premise of BONCAT is that the non-canonical amino acids (NCAA) used to track translational activity do not significantly alter cellular physiology. If the NCAA would induce changes in the metabolic state of cells, interpretation of BONCAT studies could be challenging. To address this knowledge-gap, we have used a global metabolomics analyses to assess the intracellular effects of NCAA incorporation. Two NCAA were tested: L-azidohomoalanine (AHA) and L-homopropargylglycine (HPG); L-methionine (MET) was used as a minimal stress baseline control. Liquid Chromatography Mass Spectrometry (LC-MS) and Nuclear Magnetic Resonance (NMR) were used to characterize intracellular metabolite profiles of Escherichia coli cultures, with multivariate statistical analysis using XCMS and MetaboAnalyst. Results show that doping with NCAA induces metabolic changes, however, the metabolic impact was not dramatic. A second set of experiments in which cultures were placed under mild stress to simulate real-world environmental conditions showed a more consistent and more robust perturbation. Pathways that changed include amino acid and protein synthesis, choline and betaine, and the TCA cycle. Globally, these changes were statistically minor, indicating that NCAA are unlikely to exert a significant impact on cells during incorporation. Our results are consistent with previous reports of NCAA doping under replete conditions and extend these results to bacterial growth under environmentally relevant conditions. Our work highlights the power of metabolomics studies in detecting cellular response to growth conditions and the complementarity of NMR and LCMS as omics tools.

Copper modulates sex-specific fructose hepatoxicity in nonalcoholic fatty liver disease (NALFD) Wistar rat models.

This study aimed to characterize the impact of dietary copper on the biochemical and hepatic metabolite changes associated with fructose toxicity in a Wistar rat model of fructose-induced liver disease. Twenty-four male and 24 female, 6-week-old, Wister rats were separated into four experimental dietary treatment groups (6 males and 6 females per group), as follows: (1) a control diet: containing no fructose with adequate copper (i.e., CuA/0% Fruct); (2) a diet regimen identical to the control and supplemented with 30% w/v fructose in the animals' drinking water (CuA/30% Fruct); (3) a diet identical to the control diet but deficient in copper content (CuD/0% Fruct) and (4) a diet identical to the control diet but deficient in copper content and supplemented with 30% w/v fructose in the drinking water (CuD/30% Fruct). The animals were fed the four diet regimens for 5 weeks, followed by euthanization and assessment of histology, elemental profiles and identification and quantitation of liver metabolites. Results from 1H nuclear magnetic resonance metabolomics revealed mechanistic insights into copper modulation of fructose hepatotoxicity through identification of distinct metabolic phenotypes that were highly correlated with diet and sex. This study also identified previously unknown sex-specific responses to both fructose supplementation and restricted copper intake, while the presence of adequate dietary copper promoted most pronounced fructose-induced metabolite changes.