New analytical methods focusing on polar metabolite analysis in mass spectrometry and NMR-based metabolomics.

Following in the footsteps of genomics and proteomics, metabolomics has revolutionised the way we investigate and understand biological systems. Rapid development in the last 25 years has been driven largely by technical innovations in mass spectrometry and nuclear magnetic resonance spectroscopy. However, despite the modest size of metabolomes relative to proteomes and genomes, methodological capabilities for robust, comprehensive metabolite analysis remain a major challenge. Therefore, development of new methods and techniques remains vital for progress in the field. Here, we review developments in LC-MS, GC-MS and NMR methods in the last few years that have enhanced quantitative and comprehensive metabolome coverage, highlighting the techniques involved, their technical capabilities, relative performance, and potential impact.

Dietary fibre supplementation enhances radiotherapy tumour control and alleviates intestinal radiation toxicity.

BACKGROUND: Non-toxic approaches to enhance radiotherapy outcomes are beneficial, particularly in ageing populations. Based on preclinical findings showing that high-fibre diets sensitised bladder tumours to irradiation by modifying the gut microbiota, along with clinical evidence of prebiotics enhancing anti-cancer immunity, we hypothesised that dietary fibre and its gut microbiota modification can radiosensitise tumours via secretion of metabolites and/or immunomodulation. We investigated the efficacy of high-fibre diets combined with irradiation in immunoproficient C57BL/6 mice bearing bladder cancer flank allografts. RESULT: Psyllium plus inulin significantly decreased tumour size and delayed tumour growth following irradiation compared to 0.2% cellulose and raised intratumoural CD8+ cells. Post-irradiation, tumour control positively correlated with Lachnospiraceae family abundance. Psyllium plus resistant starch radiosensitised the tumours, positively correlating with Bacteroides genus abundance and increased caecal isoferulic acid levels, associated with a favourable response in terms of tumour control. Psyllium plus inulin mitigated the acute radiation injury caused by 14 Gy. Psyllium plus inulin increased caecal acetate, butyrate and propionate levels, and psyllium alone and psyllium plus resistant starch increased acetate levels. Human gut microbiota profiles at the phylum level were generally more like mouse 0.2% cellulose profiles than high fibre profiles. CONCLUSION: These supplements may be useful in combination with radiotherapy in patients with pelvic malignancy. Video Abstract.

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.

Cone photoreceptor phosphodiesterase PDE6H inhibition regulates cancer cell growth and metabolism, replicating the dark retina response.

BACKGROUND: PDE6H encodes PDE6γ', the inhibitory subunit of the cGMP-specific phosphodiesterase 6 in cone photoreceptors. Inhibition of PDE6, which has been widely studied for its role in light transduction, increases cGMP levels. The purpose of this study is to characterise the role of PDE6H in cancer cell growth. METHODS: From an siRNA screen for 487 genes involved in metabolism, PDE6H was identified as a controller of cell cycle progression in HCT116 cells. Role of PDE6H in cancer cell growth and metabolism was studied through the effects of its depletion on levels of cell cycle controllers, mTOR effectors, metabolite levels, and metabolic energy assays. Effect of PDE6H deletion on tumour growth was also studied in a xenograft model. RESULTS: PDE6H knockout resulted in an increase of intracellular cGMP levels, as well as changes to the levels of nucleotides and key energy metabolism intermediates. PDE6H knockdown induced G1 cell cycle arrest and cell death and reduced mTORC1 signalling in cancer cell lines. Both knockdown and knockout of PDE6H resulted in the suppression of mitochondrial function. HCT116 xenografts revealed that PDE6H deletion, as well as treatment with the PDE5/6 inhibitor sildenafil, slowed down tumour growth and improved survival, while sildenafil treatment did not have an additive effect on slowing the growth of PDE6γ'-deficient tumours. CONCLUSIONS: Our results indicate that the changes in cGMP and purine pools, as well as mitochondrial function which is observed upon PDE6γ' depletion, are independent of the PKG pathway. We show that in HCT116, PDE6H deletion replicates many effects of the dark retina response and identify PDE6H as a new target in preventing cancer cell proliferation and tumour growth.

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.

N-Acyloxymethyl-phthalimides deliver genotoxic formaldehyde to human cells.

Formaldehyde is a pollutant and human metabolite that is toxic at high concentrations. Biological studies on formaldehyde are hindered by its high reactivity and volatility, which make it challenging to deliver quantitatively to cells. Here, we describe the development and validation of a set of N-acyloxymethyl-phthalimides as cell-relevant formaldehyde delivery agents. These esterase-sensitive compounds were similarly or less inhibitory to human cancer cell growth than free formaldehyde but the lead compound increased intracellular formaldehyde concentrations, increased cellular levels of thymidine derivatives (implying increased formaldehyde-mediated carbon metabolism), induced formation of cellular DNA-protein cross-links and induced cell death in pancreatic cancer cells. Overall, our N-acyloxymethyl-phthalimides and control compounds provide an accessible and broadly applicable chemical toolkit for formaldehyde biological research and have potential as cancer therapeutics.

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.

Trehalose-6-phosphate signaling regulates lateral root formation in Arabidopsis thaliana.

Plant roots explore the soil for water and nutrients, thereby determining plant fitness and agricultural yield, as well as determining ground substructure, water levels, and global carbon sequestration. The colonization of the soil requires investment of carbon and energy, but how sugar and energy signaling are integrated with root branching is unknown. Here, we show through combined genetic and chemical modulation of signaling pathways that the sugar small-molecule signal, trehalose-6-phosphate (T6P) regulates root branching through master kinases SNF1-related kinase-1 (SnRK1) and Target of Rapamycin (TOR) and with the involvement of the plant hormone auxin. Increase of T6P levels both via genetic targeting in lateral root (LR) founder cells and through light-activated release of the presignaling T6P-precursor reveals that T6P increases root branching through coordinated inhibition of SnRK1 and activation of TOR. Auxin, the master regulator of LR formation, impacts this T6P function by transcriptionally down-regulating the T6P-degrader trehalose phosphate phosphatase B in LR cells. Our results reveal a regulatory energy-balance network for LR formation that links the 'sugar signal' T6P to both SnRK1 and TOR downstream of auxin.

On the interdependence of ketone body oxidation, glycogen content, glycolysis and energy metabolism in the heart.

In heart, glucose and glycolysis are important for anaplerosis and potentially therefore for d-ß-hydroxybutyrate (ßHB) oxidation. As a glucose store, glycogen may also furnish anaplerosis. We determined the effects of glycogen content on ßHB oxidation and glycolytic rates, and their downstream effects on energetics, in the isolated rat heart. High glycogen (HG) and low glycogen (LG) containing hearts were perfused with 11 mM [5-3 H]glucose and/or 4 mM [14 C]ßHB to measure glycolytic rates or ßHB oxidation, respectively, then freeze-clamped for glycogen and metabolomic analyses. Free cytosolic [NAD+ ]/[NADH] and mitochondrial [Q+ ]/[QH2 ] ratios were estimated using the lactate dehydrogenase and succinate dehydrogenase reaction, respectively. Phosphocreatine (PCr) and inorganic phosphate (Pi ) concentrations were measured using 31 P-nuclear magnetic resonance spectroscopy. Rates of ßHB oxidation in LG hearts were half that in HG hearts, with ßHB oxidation directly proportional to glycogen content. ßHB oxidation decreased glycolysis in all hearts. Glycogenolysis in glycogen-replete hearts perfused with ßHB alone was twice that of hearts perfused with ßHB and glucose, which had significantly higher levels of the glycolytic intermediates fructose 1,6-bisphosphate and 3-phosphoglycerate, and higher free cytosolic [NAD+ ]/[NADH]. ßHB oxidation increased the Krebs cycle intermediates citrate, 2-oxoglutarate and succinate, the total NADP/H pool, reduced mitochondrial [Q+ ]/[QH2 ], and increased the calculated free energy of ATP hydrolysis (∆GATP ). Although ßHB oxidation inhibited glycolysis, glycolytic intermediates were not depleted, and cytosolic free NAD remained oxidised. ßHB oxidation alone increased Krebs cycle intermediates, reduced mitochondrial Q and increased ∆GATP . We conclude that glycogen facilitates cardiac ßHB oxidation by anaplerosis. KEY POINTS: Ketone bodies (d-ß-hydroxybutyrate, acetoacetate) are increasingly recognised as important cardiac energetic substrates, in both healthy and diseased hearts. As 2-carbon equivalents they are cataplerotic, causing depletion of Krebs cycle intermediates; therefore their utilisation requires anaplerotic supplementation, and intra-myocardial glycogen has been suggested as a potential anaplerotic source during ketone oxidation. It is demonstrated here that cardiac glycogen does indeed provide anaplerotic substrate to facilitate ß-hydroxybutyrate oxidation in isolated perfused rat heart, and this contribution was quantified using a novel pulse-chase metabolic approach. Further, using metabolomics and 31 P-MR, it was shown that glycolytic flux from myocardial glycogen increased the heart's ability to oxidise ßHB, and ßHB oxidation increased the mitochondrial redox potential, ultimately increasing the free energy of ATP hydrolysis.

Disrupted propionate metabolism evokes transcriptional changes in the heart by increasing histone acetylation and propionylation.

Propiogenic substrates and gut bacteria produce propionate, a post-translational protein modifier. In this study, we used a mouse model of propionic acidaemia (PA) to study how disturbances to propionate metabolism result in histone modifications and changes to gene expression that affect cardiac function. Plasma propionate surrogates were raised in PA mice, but female hearts manifested more profound changes in acyl-CoAs, histone propionylation and acetylation, and transcription. These resulted in moderate diastolic dysfunction with raised diastolic Ca2+, expanded end-systolic ventricular volume and reduced stroke volume. Propionate was traced to histone H3 propionylation and caused increased acetylation genome-wide, including at promoters of Pde9a and Mme, genes related to contractile dysfunction through downscaled cGMP signaling. The less severe phenotype in male hearts correlated with ß-alanine buildup. Raising ß-alanine in cultured myocytes treated with propionate reduced propionyl-CoA levels, indicating a mechanistic relationship. Thus, we linked perturbed propionate metabolism to epigenetic changes that impact cardiac function.

Role of Oxylipins in the Inflammatory-Related Diseases NAFLD, Obesity, and Type 2 Diabetes.

Oxygenated polyunsaturated fatty acids (oxylipins) are bioactive molecules established as important mediators during inflammation. Different classes of oxylipins have been found to have opposite effects, e.g., pro-inflammatory prostaglandins and anti-inflammatory resolvins. Production of the different classes of oxylipins occurs during distinct stages of development and resolution of inflammation. Chronic inflammation is involved in the progression of many pathophysiological conditions and diseases such as non-alcoholic fatty liver disease, insulin resistance, diabetes, and obesity. Determining oxylipin profiles before, during, and after inflammatory-related diseases could provide clues to the onset, development, and prevention of detrimental conditions. This review focusses on recent developments in our understanding of the role of oxylipins in inflammatory disease, and outlines novel technological advancements and approaches to study their action.

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.

Unlocking the organic residues preserved in the corrosion from the Pewsey Hoard vessels.

The characterization of archaeological metal corrosion has traditionally been limited to the identification of inorganic compounds usually by X-ray diffraction (XRD), thought to result from the interaction between the metal object and the deposition environment. The discovery of a hoard of Late Roman copper-alloy vessels in Wiltshire, UK presented an unique opportunity to adopt a multi-analytical approach to characterize corrosion combining XRD with Fourier-transform infrared (FTIR) and gas chromatography with quadrupole time-of-flight mass spectrometry using a thermal separation probe (GC-QTOF-MS with TSP). This approach revealed organic compounds potentially historical preserved within crystalline inorganic matrices. It has been known for some time that ceramics can harbour organic residues, which provide crucial evidence about the use of these vessels in the past. Our results confirms that similar residues appear to survive in metal corrosion thus extending the potential for identification of biomaterials used in the past.

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.

The influence of pesticides on the corrosion of a Roman bowl excavated in Kent, UK.

We analysed corrosion from a copper bowl dating from the Roman period (43-410 AD) found in a farm in Kent, UK. Despite its relatively good condition, the interior and exterior surface of the object had areas of deterioration containing green and brown-coloured corrosion which were sampled for characterization by a multi-analytical protocol. Basic copper chlorides atacamite and paratacamite were identified in the context of mineral phases along with chlorobenzenes in the green corrosion. Chlorobenzenes are common soil contaminants in rural areas from the use of pesticides, many of which were banned more than 50 years ago. Here we show that their presence is associated with accelerated corrosion, and this provides a threat to the preservation of archaeological metal objects in the ground.

Resistance to the isocitrate dehydrogenase 1 mutant inhibitor ivosidenib can be overcome by alternative dimer-interface binding inhibitors.

Ivosidenib, an inhibitor of isocitrate dehydrogenase 1 (IDH1) R132C and R132H variants, is approved for the treatment of acute myeloid leukaemia (AML). Resistance to ivosidenib due to a second site mutation of IDH1 R132C, leading to IDH1 R132C/S280F, has emerged. We describe biochemical, crystallographic, and cellular studies on the IDH1 R132C/S280F and R132H/S280F variants that inform on the mechanism of second-site resistance, which involves both modulation of inhibitor binding at the IDH1 dimer-interface and alteration of kinetic properties, which enable more efficient 2-HG production relative to IDH1 R132C and IDH1 R132H. Importantly, the biochemical and cellular results demonstrate that it should be possible to overcome S280F mediated resistance in AML patients by using alternative inhibitors, including some presently in phase 2 clinical trials.

Polyphenols: Bioavailability, Microbiome Interactions and Cellular Effects on Health in Humans and Animals.

Polyphenolic compounds have a variety of functions in plants including protecting them from a range of abiotic and biotic stresses such as pathogenic infections, ionising radiation and as signalling molecules. They are common constituents of human and animal diets, undergoing extensive metabolism by gut microbiota in many cases prior to entering circulation. They are linked to a range of positive health effects, including anti-oxidant, anti-inflammatory, antibiotic and disease-specific activities but the relationships between polyphenol bio-transformation products and their interactions in vivo are less well understood. Here we review the state of knowledge in this area, specifically what happens to dietary polyphenols after ingestion and how this is linked to health effects in humans and animals; paying particular attention to farm animals and pigs. We focus on the chemical transformation of polyphenols after ingestion, through microbial transformation, conjugation, absorption, entry into circulation and uptake by cells and tissues, focusing on recent findings in relation to bone. We review what is known about how these processes affect polyphenol bioactivity, highlighting gaps in knowledge. The implications of extending the use of polyphenols to treat specific pathogenic infections and other illnesses is explored.

Liver glycogen phosphorylase is upregulated in glioblastoma and provides a metabolic vulnerability to high dose radiation.

Channelling of glucose via glycogen, known as the glycogen shunt, may play an important role in the metabolism of brain tumours, especially in hypoxic conditions. We aimed to dissect the role of glycogen degradation in glioblastoma (GBM) response to ionising radiation (IR). Knockdown of the glycogen phosphorylase liver isoform (PYGL), but not the brain isoform (PYGB), decreased clonogenic growth and survival of GBM cell lines and sensitised them to IR doses of 10-12 Gy. Two to five days after IR exposure of PYGL knockdown GBM cells, mitotic catastrophy and a giant multinucleated cell morphology with senescence-like phenotype developed. The basal levels of the lysosomal enzyme alpha-acid glucosidase (GAA), essential for autolysosomal glycogen degradation, and the lipidated forms of gamma-aminobutyric acid receptor-associated protein-like (GABARAPL1 and GABARAPL2) increased in shPYGL U87MG cells, suggesting a compensatory mechanism of glycogen degradation. In response to IR, dysregulation of autophagy was shown by accumulation of the p62 and the lipidated form of GABARAPL1 and GABARAPL2 in shPYGL U87MG cells. IR increased the mitochondrial mass and the colocalisation of mitochondria with lysosomes in shPYGL cells, thereby indicating reduced mitophagy. These changes coincided with increased phosphorylation of AMP-activated protein kinase and acetyl-CoA carboxylase 2, slower ATP generation in response to glucose loading and progressive loss of oxidative phosphorylation. The resulting metabolic deficiencies affected the availability of ATP required for mitosis, resulting in the mitotic catastrophy observed in shPYGL cells following IR. PYGL mRNA and protein levels were higher in human GBM than in normal human brain tissues and high PYGL mRNA expression in GBM correlated with poor patient survival. In conclusion, we show a major new role for glycogen metabolism in GBM cancer. Inhibition of glycogen degradation sensitises GBM cells to high-dose IR indicating that PYGL is a potential novel target for the treatment of GBMs.

The effects of endogenously- and exogenously-induced hyperketonemia on exercise performance and adaptation.

Elevating blood ketones may enhance exercise capacity and modulate adaptations to exercise training; however, these effects may depend on whether hyperketonemia is induced endogenously through dietary carbohydrate restriction, or exogenously through ketone supplementation. To determine this, we compared the effects of endogenously- and exogenously-induced hyperketonemia on exercise capacity and adaptation. Trained endurance athletes undertook 6 days of laboratory based cycling ("race") whilst following either: a carbohydrate-rich control diet (n = 7; CHO); a carbohydrate-rich diet + ketone drink four-times daily (n = 7; Ex Ket); or a ketogenic diet (n = 7; End Ket). Exercise capacity was measured daily, and adaptations in exercise metabolism, exercise physiology and postprandial insulin sensitivity (via an oral glucose tolerance test) were measured before and after dietary interventions. Urinary ß-hydroxybutyrate increased by ⁓150-fold and ⁓650-fold versus CHO with Ex Ket and End Ket, respectively. Exercise capacity was increased versus pre-intervention by ~5% on race day 1 with CHO (p < 0.05), by 6%-8% on days 1, 4, and 6 (all p < 0.05) with Ex Ket and decreased by 48%-57% on all race days (all p > 0.05) with End Ket. There was an ⁓3-fold increase in fat oxidation from pre- to post-intervention (p < 0.05) with End Ket and increased perceived exercise exertion (p < 0.05). No changes in exercise substrate metabolism occurred with Ex Ket, but participants had blunted postprandial insulin sensitivity (p < 0.05). Dietary carbohydrate restriction and ketone supplementation both induce hyperketonemia; however, these are distinct physiological conditions with contrasting effects on exercise capacity and adaptation to exercise training.