New Developments in 1H NMR-linked Metabolomics: Identification of New Biomarkers for the Metabolomic Classification of Niemann-Pick Disease, Type C1, and its Response to Treatment

NMR-linked metabolomics analysis was employed to investigate urinary and human plasma profiles collected from Niemann Pick type C1 disease patients (NP-C1), in addition to aqueous extracts of liver samples of an NP-C1 mouse model. NP-C1 is a lysosomal storage disorder caused by mutations in the lysosomal proteins NPC1 and NPC2, which are involved in lysosomal cholesterol trafficking. NP-C1 disease is a fatal genetic disorder, characterised by neurodegeneration and hepatic damage. Miglustat (MGS) is the only approved drug for this disease, and consequently, plasma and urine samples collected from MGS-treated patients were also investigated. The ability of 1H NMR analysis to detect a wide range of metabolites simultaneously served to characterize the metabolic profiles of urine, plasma and hepatic tissue samples investigated in order to perform linked multivariate analysis (MVA). Additionally, MGS was identified in urine samples collected from NP-C1 treated patients. MVA employing both parametric and machine learning-based techniques was conducted to classify samples according to their disease status, and also to seek biomarkers that could aid in the diagnosis and/or prognosis of the disease. Moreover, a new technique was introduced in a metabolomics context, Correlated Component Regression (CCR), and the suitability of Random Forests (RFs) for variable selection was also explored. We were able to differentiate urine samples collected from NP-C1 patients from those collected from heterozygous controls, and also propose several metabolites as NP-C1 urinary biomarkers such as bile acids, 2-hydroxy-3-methylbutyrate, 3-aminoisobutyrate, 5-aminovalerate, trimethylamine, methanol, creatine and quinolinate. The 1H NMR linked metabolomics study of plasma samples revealed major distinctions among the groups investigated, metabolic alterations ascribable to the disease pathology were mainly observed as changes in the lipoprotein profiles of NP-C1 patients. Hepatic tissue extracts analysed revealed major disturbances in amino acid metabolism, along with impairments in the NAD+/NADH production and redox status. Gut microbiota and bile acid metabolism were also highlighted as features altered in NP-C1 disease. CCR linked to Linear Discriminant Analysis was evaluated as a new tool for metabolomics analysis, giving accurate results when compared to alternative techniques tested. Additionally, the suitability of Random Forests and associated recursive feature elimination for variable selection in metabolomics studies was contrasted, suggesting that those strategies relying on a variable ranking to select the top features for discrimination are more suitable for metabolomics investigations than those that iteratively remove a percentage of the least effective features until the classification performance decays.Hope Against Cance

1H NMR-Linked Metabolomics Analysis of Liver from a Mouse Model of NP-C1 Disease

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link

A Multifactorial Comparison of Ternary Combinations of Essential Oils in Topical Preparations to Current Antibiotic Prescription Therapies for the Control of Acne Vulgaris-Associated Bacteria.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.Acne vulgaris, a chronic condition associated with overgrowth of Propionibacterium acnes and Staphylococcus epidermidis, is commonly treated with antibiotics. However, the emergence of antibiotic resistance has resulted in a need for alternative therapies. The aim of this study is to develop a topical preparation incorporating Essential Oils (EOs) for use against acne-associated bacteria and assess its efficacy against prescription therapies Dalacin T and Stiemycin. Antimicrobial screening of rosewood, clove bud and litsea EOs was conducted and interactions between binary and ternary combinations determined against P. acnes and S. epidermidis (type and clinical isolates) using Minimum Inhibitory Concentrations (MICs) and Fractional Inhibitory Concentrations (FICs). The EOs were characterised by both GC-MS and NMR. A combination of 0.53 mg/ml litsea, 0.11 mg/ml rosewood and 0.11 mg/ml clove bud were formulated into herbal distillates and compared to Dalacin T and Stiemycin against antibiotic sensitive and resistant isolates (erythromycin). The distillate with EO had synergistic activity against P. acnes (7log10 reduction) and indifferent activity against S. epidermidis, (6log10 reduction), antimicrobial activity was either significantly (p ≤ 0.05) more antimicrobial or equivalent to that of Dalacin T and Stiemycin. This formulation may serve as a valuable alternative for the control of acne vulgaris-associated bacteria

Methylphenidate alters monoaminergic and metabolic pathways in the cerebellum of adolescent rats

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI linkAbnormalities in the cerebellar circuitry have been suggested to contribute to some of the symptoms associated with attention deficit hyperactivity disorder (ADHD). The psychostimulant methylphenidate (MPH) is the major drug for treating this condition. Here, the effects of acute (2.0 mg/kg and 5.0 mg/kg) and chronic (2.0 mg/kg, twice daily for 15 days) MPH treatments were investigated in adolescent (35-40 days old) rats on monoaminergic and metabolic markers in the cerebellum. Data acquired indicates that acute MPH treatment (2.0 mg/kg) decreased cerebellar vesicular monoamine transporter (VMAT2) density, while chronic treatment caused an increase. In contrast, protein levels of tyrosine hydroxylase (TH) and the dopamine D1 receptor were not significantly altered by neither acute nor chronic MPH treatment. In addition, while chronic but not acute MPH treatment significantly enhanced dopamine turnover (DOPAC/dopamine) in the cerebellum, levels of dopamine and homovanillic acid (HVA) were not altered. Acute MPH (5.0 mg/kg) significantly modified levels of a range of cerebellar metabolites with similar trends also detected for the lower dose (2.0 mg/kg). In this regard, acute MPH tended to decrease cerebellar metabolites associated with energy consumption and excitatory neurotransmission including glutamate, glutamine, N-acetyl aspartate, and inosine. Conversely, levels of some metabolites associated with inhibitory neurotransmission, including GABA and glycine were reduced by acute (5.0 mg/kg) MPH, together with acetate, aspartate and hypoxanthine. In conclusion, this study demonstrated that MPH alters cerebellar biochemistry, and that this effect depends on both dose and duration of treatment. The therapeutic significance of these results requires further investigation

1H NMR-based metabolomics reveals neurochemical alterations in the brain of adolescent rats following acute methylphenidate administration

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link.The psychostimulant methylphenidate (MPH) is increasingly used in the treatment of attention deficit hyperactivity disorder (ADHD). While there is little evidence for common brain pathology in ADHD, some studies suggest a right hemisphere dysfunction among people diagnosed with the condition. However, in spite of the high usage of MPH in children and adolescents, its mechanism of action is poorly understood. Given that MPH blocks the neuronal transporters for dopamine and noradrenaline, most research into the effects of MPH on the brain has largely focused on these two monoamine neurotransmitter systems. Interestingly, recent studies have demonstrated metabolic changes in the brain of ADHD patients, but the impact of MPH on endogenous brain metabolites remains unclear. In this study, a proton nuclear magnetic resonance (1H NMR)-based metabolomics approach was employed to investigate the effects of MPH on brain biomolecules. Adolescent male Sprague Dawley rats were injected intraperitoneally with MPH (5.0 mg/kg) or saline (1.0 ml/kg), and cerebral extracts from the left and right hemispheres were analysed. A total of 22 variables (representing 13 distinct metabolites) were significantly increased in the MPH-treated samples relative to the saline-treated controls. The upregulated metabolites included: amino acid neurotransmitters such as GABA, glutamate and aspartate; large neutral amino acids (LNAA), including the aromatic amino acids (AAA) tyrosine and phenylalanine, both of which are involved in the metabolism of dopamine and noradrenaline; and metabolites associated with energy and cell membrane dynamics, such as creatine and myo-inositol. No significant differences in metabolite concentrations were found between the left and right cerebral hemispheres. These findings provide new insights into the mechanisms of action of the anti-ADHD drug MPH

Antimicrobial Activity of Ternary Essential Oil Mixtures in Topical Cosmetic Preparations Against Acne Vulgaris-Associated Bacteria.

Acne vulgaris is a chronic skin condition associated with overgrowth of Propionibacterium acnes and Staphylococcus epidermidis. Antibiotics are commonly prescribed to control acne; however, resistance has emerged, indicating the need for new antimicrobial therapies1. Several essential oils (EOs) are antimicrobial, including against antibiotic-resistant isolates, indicating that antimicrobial formulations may be developed using EOs2. The aim of this study was to develop a topical preparation incorporating EOs to control acne-associated bacteria, and compare its efficacy with the topical antibiotic formulations Dalacin T® and Stiemycin®. The antimicrobial activity of litsea (Litsea cubeba), rosewood (Aniba rosaeadora) and clove bud (Syzigum aromaticum) EOs was identified using the Kirby-Bauer disc diffusion method3 and the minimum inhibitory concentrations (MICs) were determined using a broth microdilution assay3.The MICs of clove bud and rosewood were 1.38 mg/ml against S. epidermidis and P. acnes. The MIC of litsea was 1.11 mg/mL against S. epidermidis and 2.23 mg/mL against P. acnes. The antimicrobial activity of a ternary combination of these EOs was further investigated using the checkerboard method with calculation of fractional inhibitory concentration index (FICI)4. Synergy was observed against both test species (FICI < 0.5). The EOs were characterised by both GC-MS and NMR. The antimicrobial efficacy of a cosmetic preparation containing the ternary EO combination was then compared to that of the current prescription medicines Dalacin T® and Stiemycin®. A combination of 0.53 mg/mL litsea, 0.11 mg/mL rosewood and 0.11 mg/mL clove bud EOs was formulated into a neroli herbal distillate with 0.5% (v/v) ethanol. Survival of P. acnes and S. epidermidis (types and clinical isolates) after a 24 h contact with the formulations was determined and compared to Dalacin T® and Stiemycin®. The formulation completely inhibited P. acnes (7 log10 reduction) and S. epidermidis (6 log10 reduction), and was equivalent in activity to Dalacin T® and Stiemycin®, including erythromycin-resistant isolates. The EO combination alone was either more antimicrobial (p ≤ 0.05) or equivalent in activity to the active ingredients of Dalacin T® and Stiemycin® (10 mg/mL clindamycin phosphate and 20 mg/mL erythromycin, respectively). The formulation may be useful as an alternative to antibiotic preparations for the control of acne-associated bacteria

Detection of Metabolic Changes Induced via Drug Treatments in Live Cancer Cells and Tissue Using Raman Imaging Microscopy

Isocitrate dehydrogenase 1 (IDH1) mutations in gliomas, fibrosarcoma, and other cancers leads to a novel metabolite, D-2-hydroxyglutarate, which is proposed to cause tumorigenesis. The production of this metabolite also causes vulnerabilities in cellular metabolism, such as lowering NADPH levels. To exploit this vulnerability, we treated glioma and fibrosarcoma cells that harbor an IDH1 mutation with an inhibitor of nicotinamide adenine dinucleotide (NAD+) salvage pathway, FK866, and observed decreased viability in these cells. To understand the mechanism of action by which the inhibitor FK866 works, we used Raman imaging microscopy and identified that proteins and lipids are decreased upon treatment with the drug. Raman imaging showed a different distribution of lipids throughout the cell in the presence of the drug compared with the untreated cells. We employed nuclear magnetic resonance NMR spectroscopy and mass spectrometry to identify the classes of lipids altered. Our combined analyses point to a decrease in cell division due to loss of lipid content that contributes to membrane formation in the in vitro setting. However, the FK866 drug did not have the same potency in vivo. The use of Raman imaging microscopy indicated an opposite trend of lipid distribution in the tissue collected from treated versus untreated mice when compared with the cells. These results demonstrate the role of Raman imaging microscopy to identify and quantify metabolic changes in cancer cells and tissue

Metabolic Landscape of a Genetically Engineered Mouse Model of IDH1 Mutant Glioma

Understanding the metabolic reprogramming of aggressive brain tumors has potential applications for therapeutics as well as imaging biomarkers. However, little is known about the nutrient requirements of isocitrate dehydrogenase 1 (IDH1) mutant gliomas. The IDH1 mutation involves the acquisition of a neomorphic enzymatic activity which generates D-2-hydroxyglutarate from &alpha;-ketoglutarate. In order to gain insight into the metabolism of these malignant brain tumors, we conducted metabolic profiling of the orthotopic tumor and the contralateral regions for the mouse model of IDH1 mutant glioma; as well as to examine the utilization of glucose and glutamine in supplying major metabolic pathways such as glycolysis and tricarboxylic acid (TCA). We also revealed that the main substrate of 2-hydroxyglutarate is glutamine in this model, and how this re-routing impairs its utilization in the TCA. Our 13C tracing analysis, along with hyperpolarized magnetic resonance experiments, revealed an active glycolytic pathway similar in both regions (tumor and contralateral) of the brain. Therefore, we describe the reprogramming of the central carbon metabolism associated with the IDH1 mutation in a genetically engineered mouse model which reflects the tumor biology encountered in glioma patients