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.

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.

Innovative method for rapid detection of falsified COVID-19 vaccines through unopened vials using handheld Spatially Offset Raman Spectroscopy (SORS).

Preventing, detecting, and responding to substandard and falsified vaccines is of critical importance for ensuring the safety, efficacy, and public trust in vaccines. This is of heightened importance in context of public health crisis, such as the COVID-19 pandemic, in which extreme world-wide shortages of vaccines provided a fertile ground for exploitation by falsifiers. Here, a proof-of-concept study explored the feasibility of using a handheld Spatially Offset Raman Spectroscopy (SORS) device to authenticate COVID-19 vaccines through rapid analysis of unopened vaccine vials. The results show that SORS can verify the chemical identity of dominant excipients non-invasively through vaccine vial walls. The ability of SORS to identify potentially falsified COVID-19 vaccines was demonstrated by measurement of surrogates for falsified vaccines contained in vaccine vials. In all cases studied, the SORS technique was able to differentiate between surrogate samples from the genuine COVISHIELD™ vaccine. The genuine vaccines tested included samples from six batches across two manufacturing sites to account for any potential variations between batches or manufacturing sites. Batch and manufacturing site variations were insignificant. In conjunction with existing security features, for example on labels and packaging, SORS provided an intrinsic molecular fingerprint of the dominant excipients of the vaccines. The technique could be extended to other COVID-19 and non-COVID-19 vaccines, as well as other liquid medicines. As handheld and portable SORS devices are commercially available and widely used for other purposes, such as airport security, they are rapidly deployable non-invasive screening tools for vaccine authentication.

Metformin maintains intrahepatic triglyceride content through increased hepatic de novo lipogenesis.

OBJECTIVE: Metformin is a first-line pharmacotherapy in the treatment of type 2 diabetes, a condition closely associated with non-alcoholic fatty liver disease (NAFLD). Although metformin promotes weight loss and improves insulin sensitivity, its effect on intrahepatic triglyceride (IHTG) remains unclear. We investigated the effect of metformin on IHTG, hepatic de novo lipogenesis (DNL), and fatty acid (FA) oxidation in vivo in humans. DESIGN AND METHODS: Metabolic investigations, using stable-isotope tracers, were performed in ten insulin-resistant, overweight/obese human participants with NAFLD who were treatment naïve before and after 12 weeks of metformin treatment. The effect of metformin on markers of s.c. adipose tissue FA metabolism and function, along with the plasma metabolome, was investigated. RESULTS: Twelve weeks of treatment with metformin resulted in a significant reduction in body weight and improved insulin sensitivity, but IHTG content and FA oxidation remained unchanged. Metformin treatment was associated with a significant decrease in VLDL-triglyceride (TG) concentrations and a significant increase in the relative contribution of DNL-derived FAs to VLDL-TG. There were subtle and relatively few changes in s.c. adipose tissue FA metabolism and the plasma metabolome with metformin treatment. CONCLUSIONS: We demonstrate the mechanisms of action of metformin whereby it improves insulin sensitivity and promotes weight loss, without improvement in IHTG; these observations are partly explained through increased hepatic DNL and a lack of change in FA oxidation.

Altered glycolysis triggers impaired mitochondrial metabolism and mTORC1 activation in diabetic ß-cells.

Chronic hyperglycaemia causes a dramatic decrease in mitochondrial metabolism and insulin content in pancreatic ß-cells. This underlies the progressive decline in ß-cell function in diabetes. However, the molecular mechanisms by which hyperglycaemia produces these effects remain unresolved. Using isolated islets and INS-1 cells, we show here that one or more glycolytic metabolites downstream of phosphofructokinase and upstream of GAPDH mediates the effects of chronic hyperglycemia. This metabolite stimulates marked upregulation of mTORC1 and concomitant downregulation of AMPK. Increased mTORC1 activity causes inhibition of pyruvate dehydrogenase which reduces pyruvate entry into the tricarboxylic acid cycle and partially accounts for the hyperglycaemia-induced reduction in oxidative phosphorylation and insulin secretion. In addition, hyperglycaemia (or diabetes) dramatically inhibits GAPDH activity, thereby impairing glucose metabolism. Our data also reveal that restricting glucose metabolism during hyperglycaemia prevents these changes and thus may be of therapeutic benefit. In summary, we have identified a pathway by which chronic hyperglycaemia reduces ß-cell function.

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.

3-Bromopyruvate-mediated MCT1-dependent metabolic perturbation sensitizes triple negative breast cancer cells to ionizing radiation.

BACKGROUND: Triple negative breast cancer (TNBC) poses a serious clinical challenge as it is an aggressive form of the disease that lacks estrogen receptor, progesterone receptor, and ERBB2 (formerly HER2) gene amplification, which limits the treatment options. The Warburg phenotype of upregulated glycolysis in the presence of oxygen has been shown to be prevalent in TNBC. Elevated glycolysis satisfies the energy requirements of cancer cells, contributes to resistance to treatment by maintaining redox homeostasis and generating nucleotide precursors required for cell proliferation and DNA repair. Expression of the monocarboxylate transporter 1 (MCT1), which is responsible for the bidirectional transport of lactate, correlates with an aggressive phenotype and poor outcome in several cancer types, including breast cancer. In this study, 3-bromopyruvate (3BP), a lactate/pyruvate analog, was used to selectively target TNBC cells that express MCT1. METHODS: The cytotoxicity of 3BP was tested in MTT assays using human TNBC cell lines: BT20 (MCT1+/MCT4-), MDA-MB-23 (MCT1-/MCT4+), and BT20 in which MCT1 was knocked down (siMCT1-BT20). The metabolite profile of 3BP-treated and 3BP-untreated cells was investigated using LC-MS/MS. The extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of BT20 and MDA-MB-231 cells treated with 3BP were measured using a Seahorse XF96 extracellular flux analyzer. The impact of ionizing radiation on cell survival, alone or in combination with 3BP pre-treatment, was evaluated using clonogenic assays. RESULTS: Metabolomic analyses showed that 3BP causes inhibition of glycolysis, disturbance of redox homeostasis, decreased nucleotide synthesis, and was accompanied by a reduction in medium acidification. In addition, 3BP potentiated the cytotoxic effect of ionizing radiation, a treatment that is frequently used in the management of TNBC. CONCLUSIONS: Overall, MCT1-mediated metabolic perturbation in combination with radiotherapy is shown to be a promising strategy for the treatment of glycolytic tumors such as TNBC, overcoming the selectivity challenges of targeting glycolysis with glucose analogs.

Roles of metal ions in the selective inhibition of oncogenic variants of isocitrate dehydrogenase 1.

Cancer linked isocitrate dehydrogenase (IDH) 1 variants, notably R132H IDH1, manifest a 'gain-of-function' to reduce 2-oxoglutarate to 2-hydroxyglutarate. High-throughput screens have enabled clinically useful R132H IDH1 inhibitors, mostly allosteric binders at the dimer interface. We report investigations on roles of divalent metal ions in IDH substrate and inhibitor binding that rationalise this observation. Mg2+/Mn2+ ions enhance substrate binding to wt IDH1 and R132H IDH1, but with the former manifesting lower Mg2+/Mn2+ KMs. The isocitrate-Mg2+ complex is the preferred wt IDH1 substrate; with R132H IDH1, separate and weaker binding of 2-oxoglutarate and Mg2+ is preferred. Binding of R132H IDH1 inhibitors at the dimer interface weakens binding of active site Mg2+ complexes; their potency is affected by the Mg2+ concentration. Inhibitor selectivity for R132H IDH1 over wt IDH1 substantially arises from different stabilities of wt and R132H IDH1 substrate-Mg2+ complexes. The results reveal the importance of substrate-metal ion complexes in wt and R132H IDH1 catalysis and the basis for selective R132H IDH1 inhibition. Further studies on roles of metal ion complexes in TCA cycle and related metabolism, including from an evolutionary perspective, are of interest.

Chromatin accessibility governs the differential response of cancer and T cells to arginine starvation.

Depleting the microenvironment of important nutrients such as arginine is a key strategy for immune evasion by cancer cells. Many tumors overexpress arginase, but it is unclear how these cancers, but not T cells, tolerate arginine depletion. In this study, we show that tumor cells synthesize arginine from citrulline by upregulating argininosuccinate synthetase 1 (ASS1). Under arginine starvation, ASS1 transcription is induced by ATF4 and CEBPß binding to an enhancer within ASS1. T cells cannot induce ASS1, despite the presence of active ATF4 and CEBPß, as the gene is repressed. Arginine starvation drives global chromatin compaction and repressive histone methylation, which disrupts ATF4/CEBPß binding and target gene transcription. We find that T cell activation is impaired in arginine-depleted conditions, with significant metabolic perturbation linked to incomplete chromatin remodeling and misregulation of key genes. Our results highlight a T cell behavior mediated by nutritional stress, exploited by cancer cells to enable pathological immune evasion.

Anion-exchange chromatography mass spectrometry provides extensive coverage of primary metabolic pathways revealing altered metabolism in IDH1 mutant cells.

Altered central carbon metabolism is a hallmark of many diseases including diabetes, obesity, heart disease and cancer. Identifying metabolic changes will open opportunities for better understanding aetiological processes and identifying new diagnostic, prognostic, and therapeutic targets. Comprehensive and robust analysis of primary metabolic pathways in cells, tissues and bio-fluids, remains technically challenging. We report on the development and validation of a highly reproducible and robust untargeted method using anion-exchange tandem mass spectrometry (IC-MS) that enables analysis of 431 metabolites, providing detailed coverage of central carbon metabolism. We apply the method in an untargeted, discovery-driven workflow to investigate the metabolic effects of isocitrate dehydrogenase 1 (IDH1) mutations in glioblastoma cells. IC-MS provides comprehensive coverage of central metabolic pathways revealing significant elevation of 2-hydroxyglutarate and depletion of 2-oxoglutarate. Further analysis of the data reveals depletion in additional metabolites including previously unrecognised changes in lysine and tryptophan metabolism.

Rapid intraoperative molecular genetic classification of gliomas using Raman spectroscopy.

BACKGROUND: The molecular genetic classification of gliomas, particularly the identification of isocitrate dehydrogenase (IDH) mutations, is critical for clinical and surgical decision-making. Raman spectroscopy probes the unique molecular vibrations of a sample to accurately characterize its molecular composition. No sample processing is required allowing for rapid analysis of tissue. The aim of this study was to evaluate the ability of Raman spectroscopy to rapidly identify the common molecular genetic subtypes of diffuse glioma in the neurosurgical setting using fresh biopsy tissue. In addition, classification models were built using cryosections, formalin-fixed paraffin-embedded (FFPE) sections and LN-18 (IDH-mutated and wild-type parental cell) glioma cell lines. METHODS: Fresh tissue, straight from neurosurgical theatres, underwent Raman analysis and classification into astrocytoma, IDH-wild-type; astrocytoma, IDH-mutant; or oligodendroglioma. The genetic subtype was confirmed on a parallel section using immunohistochemistry and targeted genetic sequencing. RESULTS: Fresh tissue samples from 62 patients were collected (36 astrocytoma, IDH-wild-type; 21 astrocytoma, IDH-mutated; 5 oligodendroglioma). A principal component analysis fed linear discriminant analysis classification model demonstrated 79%-94% sensitivity and 90%-100% specificity for predicting the 3 glioma genetic subtypes. For the prediction of IDH mutation alone, the model gave 91% sensitivity and 95% specificity. Seventy-nine cryosections, 120 FFPE samples, and LN18 cells were also successfully classified. Meantime for Raman data collection was 9.5 min in the fresh tissue samples, with the process from intraoperative biopsy to genetic classification taking under 15 min. CONCLUSION: These data demonstrate that Raman spectroscopy can be used for the rapid, intraoperative, classification of gliomas into common genetic subtypes.