Aldehyde-mediated inhibition of asparagine biosynthesis has implications for diabetes and alcoholism

Patients with alcoholism and type 2 diabetes manifest altered metabolism, including elevated aldehyde levels and unusually low asparagine levels. We show that asparagine synthetase B (ASNS), the only human asparagine-forming enzyme, is inhibited by disease-relevant reactive aldehydes, including formaldehyde and acetaldehyde. Cellular studies show non-cytotoxic amounts of reactive aldehydes induce a decrease in asparagine levels. Biochemical analyses reveal inhibition results from reaction of the aldehydes with the catalytically important N-terminal cysteine of ASNS. The combined cellular and biochemical results suggest a possible mechanism underlying the low asparagine levels in alcoholism and diabetes. The results will stimulate research on the biological consequences of the reactions of aldehydes with nucleophilic residues

Repurposing rapid diagnostic tests to detect falsified vaccines in supply chains

Substandard (including degraded) and falsified (SF) vaccines are a relatively neglected issue with serious global implications for public health. This has been highlighted during the rapid and widespread rollout of COVID-19 vaccines. There has been increasing interest in devices to screen for SF non-vaccine medicines including tablets and capsules to empower inspectors and standardise surveillance. However, there has been very limited published research focussed on repurposing or developing new devices for screening for SF vaccines. To our knowledge, rapid diagnostic tests (RDTs) have not been used for this purpose but have important potential for detecting falsified vaccines. We performed a proof-in-principle study to investigate their diagnostic accuracy using a diverse range of RDT-vaccine/falsified vaccine surrogate pairs. In an initial assessment, we demonstrated the utility of four RDTs in detecting seven vaccines. Subsequently, the four RDTs were evaluated by three blinded assessors with seven vaccines and four falsified vaccines surrogates. The results provide preliminary data that RDTs could be used by multiple international organisations, national medicines regulators and vaccine manufacturers/distributors to screen for falsified vaccines in supply chains, aligned with the WHO global ‘Prevent, Detect and Respond’ strategy

Tibetan PHD2, an allele with loss-of-function properties

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, PHD2 and HIF2A. The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan PHD2 are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan PHD2 is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan Phd2 allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in p23 that abrogates the PHD2:p23 interaction. Hif2a haploinsufficiency, but not the Tibetan Phd2 allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan Phd2 allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude

Variants in ALDH1A2 reveal an anti-inflammatory role for retinoic acid and a new class of disease-modifying drugs in osteoarthritis

More than 40% of individuals will develop osteoarthritis (OA) during their lifetime, yet there are currently no licensed disease-modifying treatments for this disabling condition. Common polymorphic variants in ALDH1A2, which encodes the key enzyme for synthesis of all-trans retinoic acid (atRA), are associated with severe hand OA. Here, we sought to elucidate the biological significance of this association. We first confirmed that ALDH1A2 risk variants were associated with hand OA in the U.K. Biobank. Articular cartilage was acquired from 33 individuals with hand OA at the time of routine hand OA surgery. After stratification by genotype, RNA sequencing was performed. A reciprocal relationship between ALDH1A2 mRNA and inflammatory genes was observed. Articular cartilage injury up-regulated similar inflammatory genes by a process that we have previously termed mechanoflammation, which we believe is a primary driver of OA. Cartilage injury was also associated with a concomitant drop in atRA-inducible genes, which were used as a surrogate measure of cellular atRA concentration. Both responses to injury were reversed using talarozole, a retinoic acid metabolism blocking agent (RAMBA). Suppression of mechanoflammation by talarozole was mediated by a peroxisome proliferator–activated receptor gamma (PPARγ)–dependent mechanism. Talarozole was able to suppress mechano-inflammatory genes in articular cartilage in vivo 6 hours after mouse knee joint destabilization and reduced cartilage degradation and osteophyte formation after 26 days. These data show that boosting atRA suppresses mechanoflammation in the articular cartilage in vitro and in vivo and identifies RAMBAs as potential disease-modifying drugs for OA

Metabolomics of the second culture of LN18 IDH1 mutant and wild-type cells

These data were produced from the analysis of the second culture of LN18 cells with (MUT) and without (WT) a transduced isocitrate dehydrogenase 1 (IDH1) mutation. The data were acquired as part of the project “Investigating Isocitrate Dehydrogenase Mutations in Cancer Using New Metabolomics Methods” using multiple liquid chromatography-mass spectrometry methods. Anion-exchange chromatography-mass spectrometry (IC-MS) data were acquired on a Dionex IC-5000+ ion chromatography system hyphenated to a Thermo Fisher Scientific Exactive. Reversed-phase chromatography-mass spectrometry (RP-MS) data were acquired on a Dionex U3000 UHPLC system hyphenated to a Thermo Fisher Scientific Exactive. Derivatised RP-MS data were acquired on a Dionex U3000 UHPLC system hyphenated to a Thermo Fisher Scientific Exactive with sample derivatisation using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Data are .zip compressed to reduce file size. Data files in the .RAW format require Thermo Fisher Scientific Xcalibur software to analyse

Metabolomics of 293T IDH1 mutant and wild-type cells

These data were produced from the analysis of the 293T cells with (MUT) and without (WT) a transduced isocitrate dehydrogenase 1 (IDH1) mutation. The data were acquired as part of the project “Investigating Isocitrate Dehydrogenase Mutations in Cancer Using New Metabolomics Methods” using multiple liquid chromatography-mass spectrometry methods. Anion-exchange chromatography-mass spectrometry (IC-MS) data were acquired on a Dionex IC-5000+ ion chromatography system hyphenated to a Thermo Fisher Scientific Exactive. Reversed-phase chromatography-mass spectrometry (RP-MS) data were acquired on a Dionex U3000 UHPLC system hyphenated to a Thermo Fisher Scientific Exactive. Derivatised RP-MS data were acquired on a Waters ACQUITY UPLC System hyphenated to a Waters Xevo G2-XS QTof with sample derivatisation using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Data are .zip compressed to reduce file size. Data files in the .RAW format require Thermo Fisher Scientific Xcalibur software to analyse. Data folders in the .RAW format require Waters Masslynx software to analyse

Metabolomics of 293T wild-type cells cultured under different glucose conditions

These data were produced from the analysis of 293T wild-type (WT) cells cultured under 4.5 g/L glucose (HG) or 1.5 g/L gluose (LG) conditions. The data were acquired as part of the project “Investigating Isocitrate Dehydrogenase Mutations in Cancer Using New Metabolomics Methods” using multiple liquid chromatography-mass spectrometry methods. Derivatised RP-MS data were acquired on a Waters ACQUITY I-Class PLUS UPLC System hyphenated to a Waters Vion IMS QTof with sample derivatisation using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Data are .zip compressed to reduce file size. Data files in the .uep format require Waters Unifi software to analyse

Metabolomics of U87-MG IDH1 mutant and wild-type cells

These data were produced from the analysis of U87-MG cells with (MUT) and without (WT) a transduced isocitrate dehydrogenase 1 (IDH1) mutation. The data were acquired as part of the project “Investigating Isocitrate Dehydrogenase Mutations in Cancer Using New Metabolomics Methods” using multiple liquid chromatography-mass spectrometry methods. Anion-exchange chromatography-mass spectrometry (IC-MS) data were acquired on a Dionex IC-5000+ ion chromatography system hyphenated to a Thermo Fisher Scientific Exactive. Reversed-phase chromatography-mass spectrometry (RP-MS) data were acquired on a Waters ACQUITY UPLC System hyphenated to a Waters Xevo G2-XS QTof. Derivatised RP-MS data were acquired on a Waters ACQUITY I-Class PLUS UPLC System hyphenated to a Waters Vion IMS QTof with sample derivatisation using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Data is .zip compressed to reduce file size. Data files in the .RAW format require Thermo Fisher Scientific Xcalibur software to analyse. Data folders in the .RAW format require Waters Masslynx software to analyse. Data files in the .uep format require Waters Unifi software to analyse

Metabolomics of 2-OG treated LN18 IDH1 mutant and wild-type cells

These data were produced from the analysis of LN18 cells with (MUT) and without (WT) a transduced isocitrate dehydrogenase 1 (IDH1) mutation, treated with 10 mM 2-oxoglutarate (2-OG) or vehicle control for 24 hours. The data were acquired as part of the project “Investigating Isocitrate Dehydrogenase Mutations in Cancer Using New Metabolomics Methods” using multiple liquid chromatography-mass spectrometry methods. Anion-exchange chromatography-mass spectrometry (IC-MS) data were acquired on a Dionex IC-5000+ ion chromatography system hyphenated to a Thermo Fisher Scientific Exactive. Reversed-phase chromatography-mass spectrometry (RP-MS) data were acquired on a Dionex U3000 UHPLC system hyphenated to a Thermo Fisher Scientific Exactive. Derivatised RP-MS data were acquired on a Dionex U3000 UHPLC system hyphenated to a Thermo Fisher Scientific Exactive with sample derivatisation using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Data are .zip compressed to reduce file size. Data files in the .RAW format require Thermo Fisher Scientific Xcalibur software to analyse

Investigating isocitrate dehydrogenase mutations in cancer using new metabolomics methods

Altered cellular metabolism, as a result of oncogenic mutations, has been observed in a range of cancer types. A detailed understanding of how cancers modify cellular metabolism, which includes changes to the genome, proteome and metabolome, is therefore integral for the development of effective and targeted therapeutic treatments. Isocitrate dehydrogenase mutations lead to a change of enzyme function, which causes the production of high intracellular concentrations of 2 hydroxyglutarate, and are found in a range of cancer types including leukaemia, chondrosarcoma, T cell lymphoma and glioma. Although a range of epigenetic and metabolic effects of isocitrate dehydrogenase 1 and 2 mutations have been identified, which suggest a pro oncogenic role, the tumorigenic mechanism is not well understood. Over 70 % of low grade glioma (grade 2 and 3) in humans have isocitrate dehydrogenase 1 or 2 mutations, and have one of the lowest 5 year survival rates of all cancer types. Consequently, there is a need for better diagnostic and therapeutic approaches. Small molecule inhibitors, which are specific to isocitrate dehydrogenase mutations and reduce the abundance of 2 hydroxyglutarate, have been developed; however, resistance to treatment has since been reported, including the manifestation of second site mutations. Increased understanding of the metabolic changes associated with isocitrate dehydrogenase mutations has the potential to improve therapeutic efficacy, and elucidate new therapeutic pathways. Metabolomics, the comprehensive analysis and identification of low molecular weight compounds in biological systems, allows the discovery of metabolic adaptations to disease and changes in environment, enabling hypothesis generation and directing further investigations. Untargeted metabolomics is typically used for the identification of biomarkers, in a discovery-led approach using statistical analysis of experimental groups. A challenge for untargeted metabolomics is the comprehensive analysis of all metabolites present in a biological sample, due to the range of chemical functionalities and matrix complexity. Analysis of polar and ionic metabolites, especially those which represent central carbon metabolism, are not well characterised by the most common hyphenated analytical methods including reversed phase chromatography, ion pair chromatography and hydrophobic interaction liquid chromatography, coupled to mass spectrometry. Development of selective eluent suppression technology has enabled the hyphenation of conventional ion chromatography to mass spectrometry, providing a potentially effective method for the analysis of polar metabolites. Anion exchange chromatography has recently been applied to targeted analysis, demonstrating reproducible analysis of some polar and ionic metabolites. Comprehensive untargeted metabolomics using ion chromatography mass spectrometry has not previously been explored to any significant extent. In this thesis, an in depth study of the metabolic effects associated an isocitrate dehydrogenase 1 mutation in cancer cells will be undertaken, which will include the development of new anion exchange chromatography and ion mobility spectrometry approaches coupled to existing state of the art mass spectrometry. The aims are to both develop and validate new methods, and use these to comprehensively explore how metabolism is altered in association with an isocitrate dehydrogenase 1 mutation. Human glioblastoma and embryonic kidney derived cell lines with and without a transduced isocitrate dehydrogenase 1 mutation will be used as model systems. It is demonstrated that anion exchange chromatography mass spectrometry provides increased sensitivity and comprehensive coverage of central carbon metabolites compared with an existing hydrophobic interaction liquid chromatography mass spectrometry method. A new ion mobility spectrometry mass spectrometry method is also developed and used for the analysis of derivatised samples, offering improved sensitivity for metabolites with primary and secondary amine functionality, and increasing confidence in identification using collisional cross section measurements. These analyses revealed that lysine and tryptophan degradation products are altered in isocitrate dehydrogenase 1 mutant cells and the possibility for inhibition of 2 oxoglutate dependent pathways by 2 hydroxyglutarate is explored. Treatment of isocitrate dehydrogenase 1 mutant cells with 2 oxoglutarate and 2 hydroxyglutarate is investigated and the results suggest additional regulatory metabolic effects in isocitrate dehydrogenase 1 mutant cells, beyond the enrichment of 2 hydroxyglutarate and the depletion of 2 oxoglutarate. The relationship between an isocitrate dehydrogenase 1 mutation and the concentration of 2 hydroxyglutarate and 2 oxoglutarate, in transduced cell lines, demonstrated the potential for metabolic therapies, in combination with existing pharmacological treatment. The alteration of immunosuppressive metabolites such as kynurenine also provides a possible explanation for different median survival times of patients with isocitrate dehydrogenase mutation positive and wild type tumours, and their distinct responses to treatment. Anion exchange chromatography mass spectrometry and ion mobility spectrometry mass spectrometry were both shown to be robust and sensitive methods for untargeted analysis of cell extracts and provide benefits, beyond current methods, for hypothesis driven research. The sensitivity of isocitrate dehydrogenase 1 mutant cells to changes in redox state and 2 oxoglutarate concentration elucidates new targets for combination therapies, to develop more selective and effective treatments. New potential biomarkers are also suggested for clinical classification of tumours with isocitrate dehydrogenase 1 mutations.</p