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Cerebral creatine deficiency syndromes (CCDS) are disorders affecting creatine synthesis or transport. Several methods have been developed to measure creatine and guanidinoacetate (GAA) in different body fluids including methods based on gas chromatography-mass spectrometry (GC-MS) and High-pressure liquid chromatography mass spectrometry (HPLC-MS). The diagnosis of CCDS is then confirmed by sequencing of creatine biosynthesis genes guanidinoacetate methyltransferase (GAMT) and Arginine: glycine amidinotransferase (GATM) and creatine transporter gene solute carrier family 6 member 8 (SLC6A8) or by functional enzymatic assay. The aim of the current study was to find the most reliable and accurate screening method for CCDS by comparing methods using Nuclear Magnetic Resonance spectroscopy (NMR), GC-MS and HPLC-MS. Additionally, this study was performed to estimate the prevalence of CCDS in a cohort of Egyptian patients and potentially to discover novel variants.The study was conducted on 150 subjects with clinical signs and symptoms consistent with CCDS. Metabolic profiling of urine samples was performed using three techniques: 1) GC-MS 2) Ultra high-pressure (or performance) liquid chromatography - Tandem Mass Spectrometry (UHPLC- MS/MS) and 3) NMR.The linearity of peak areas for creatine and GAA by UHPLC-MS/MS and NMR covered and exceeded the ranges normally found in urine. The limit of quantification and the inter-day precision results for creatine and GAA were more robust by UHPLC-MS/MS than NMR. Ten cases were identified as being positive for CCDS by our analytical approaches and underwent next generation sequencing (NGS) for GAMT, GATM and SLC6A8 genes. NGS was performed and confirmed one patient with one likely Pathogenic variant in GAMT gene: (NC_000019.10:g.1401317C G, NP_000147.1:p.Ala54Pro). Additionally, we describe four novel intronic variants in the GATM gene: c.1043-357del and c.1043-357_1043-356insT, and were predicted to activate cryptic acceptor site with potential alteration of splicing, c.979-227G A was found to significantly alter the Exon Splice Enhancer (ESE) xon Splice Silencer (ESS) motifs ratio and c.1042 + 262del which was found to have no implications on splicing.Both UHPLC-MS/MS and NMR spectroscopy are comparable to GC-MS in screening for CCDS. Nonetheless, the UHPLC-MS/MS method had better performance than NMR spectroscopy. Additionally, Sequencing of the full length of GATM, GAMT, and SLC6A8 genes is needed to identify intronic variants that could cause CCDS via affecting splice sites.

BACKGROUND: Sodium–glucose co-transporter 2 inhibitors (SGLT2i) have emerged as a paramount treatment for patients with heart failure (HF), irrespective of underlying reduced or preserved ejection fraction. However, a definite cardiac mechanism of action remains elusive. Derangements in myocardial energy metabolism are detectable in all HF phenotypes, and it was proposed that SGLT2i may improve energy production. The authors aimed to investigate whether treatment with empagliflozin leads to changes in myocardial energetics, serum metabolomics, and cardiorespiratory fitness. METHODS: EMPA-VISION is a prospective, randomized, double-blind, placebo-controlled, mechanistic trial that enrolled 72 symptomatic patients with chronic HF with reduced ejection fraction (HFrEF; n=36; left ventricular ejection fraction ≤40%; New York Heart Association class ≥II; NT-proBNP [N-terminal pro-B-type natriuretic peptide] ≥125 pg/mL) and HF with preserved ejection fraction (HFpEF; n=36; left ventricular ejection fraction ≥50%; New York Heart Association class ≥II; NT-proBNP ≥125 pg/mL). Patients were stratified into respective cohorts (HFrEF versus HFpEF) and randomly assigned to empagliflozin (10 mg; n=35: 17 HFrEF and 18 HFpEF) or placebo (n=37: 19 HFrEF and 18 HFpEF) once daily for 12 weeks. The primary end point was a change in the cardiac phosphocreatine:ATP ratio (PCr:ATP) from baseline to week 12, determined by phosphorus magnetic resonance spectroscopy at rest and during peak dobutamine stress (65% of age-maximum heart rate). Mass spectrometry on a targeted set of 19 metabolites was performed at baseline and after treatment. Other exploratory end points were investigated. RESULTS: Empagliflozin treatment did not change cardiac energetics (ie, PCr:ATP) at rest in HFrEF (adjusted mean treatment difference [empagliflozin – placebo], –0.25 [95% CI, –0.58 to 0.09]; P =0.14) or HFpEF (adjusted mean treatment difference, –0.16 [95% CI, –0.60 to 0.29]; P =0.47]. Likewise, there were no changes in PCr:ATP during dobutamine stress in HFrEF (adjusted mean treatment difference, –0.13 [95% CI, –0.35 to 0.09]; P =0.23) or HFpEF (adjusted mean treatment difference, –0.22 [95% CI, –0.66 to 0.23]; P =0.32). No changes in serum metabolomics or levels of circulating ketone bodies were observed. CONCLUSIONS: In patients with either HFrEF or HFpEF, treatment with 10 mg of empagliflozin once daily for 12 weeks did not improve cardiac energetics or change circulating serum metabolites associated with energy metabolism when compared with placebo. Based on our results, it is unlikely that enhancing cardiac energy metabolism mediates the beneficial effects of SGLT2i in HF. REGISTRATION: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT03332212.

Relative abundances of free fatty acids, phosphatidylglycerols and phosphatidylinositols in tumour compared to normal lung tissue.

Figure legends for supplemental Figures S1-S6.

MYC-mediated pathogenesis in lung cancer continues to attract interest for new therapeutic strategies. In this study, we describe a transgenic mouse model of KRAS-driven lung adenocarcinoma that affords reversible activation of MYC, used here as a tool for lipidomic profiling of MYC-dependent lung tumors formed in this model. Advanced mass spectrometric imaging and surface analysis techniques were used to characterize the spatial and temporal changes in lipid composition in lung tissue. We found that normal lung tissue was characterized predominantly by saturated phosphatidylcholines and phosphatidylglycerols, which are major lipid components of pulmonary surfactant. In contrast, tumor tissues displayed an increase in phosphatidylinositols and arachidonate-containing phospholipids that can serve as signaling precursors. Deactivating MYC resulted in a rapid and dramatic decrease in arachidonic acid and its eicosanoid metabolites. In tumors with high levels of MYC, we found an increase in cytosolic phospholipase A2 (cPLA2) activity with a preferential release of membrane-bound arachidonic acid, stimulating the lipoxygenase (LOX) and COX pathways also amplified by MYC at the level of gene expression. Deactivating MYC lowered cPLA2 activity along with COX2 and 5-LOX mRNA levels. Notably, inhibiting the COX/5-LOX pathways in vivo reduced tumor burden in a manner associated with reduced cell proliferation. Taken together, our results show how MYC drives the production of specific eicosanoids critical for lung cancer cell survival and proliferation, with possible implications for the use of COX and LOX pathway inhibitors for lung cancer therapy. Cancer Res; 76(16); 4608–18. ©2016 AACR.

LC-MS/MS method details for eicosanoid analysis (Tables S1 and S2); Lipid ID by accurate mass and tandem MS (Tables S3 and S4); Fold change for eicosanoid-related species with MYC activated (Table S5).

Supplementary Methods from Novel Theranostic Opportunities Offered by Characterization of Altered Membrane Lipid Metabolism in Breast Cancer Progression

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Supplementary Table 1 from Novel Theranostic Opportunities Offered by Characterization of Altered Membrane Lipid Metabolism in Breast Cancer Progression

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BackgroundTwo-thirds of people over 65 have two or more underlying chronic conditions. Patients with multimorbidities are likely to classify as frail and have worse outcomes after cardiac surgery. We hypothesized that metabolite and transcript profiles could identify multimorbidity-specific mechanisms for clinical interventions.Methods and ResultsThe multimorbidity was defined as two or more coexisting chronic conditions. Analysis of the metabolome was performed in 30 sequential patients. Measurements of transcriptome and metabolites involved in energy production were performed in 53 and 57 sequential patients, respectively. Mitochondrial function in circulating monocytes was performed in 63 sequential patients. Our analysis distinguished three major processes that are affected by multimorbidity: innate immune response, DNA damage and associated epigenetic changes, and mitochondrial energy production. The innate immune response was upregulated in multimorbidity and most of the included comorbidities. The DNA damage, epigenetic changes and aspects of mitochondrial function were specific for multimorbidity. Histone 2B, its ubiquitination enzymes and AKT3 were upregulated in the multimorbid group suggesting senescence-like changes in gene expression. That was confirmed by the detection of senescence-associated secretory phenotype analytes, IL-1β, its receptor and fractalkine that increased with the number of accumulating comorbidities. DNA damage was confirmed by independent immunohistochemistry experiments, which also identified nucleolar instability as more prominent in the multimorbid myocardium. The nucleolar stress is potentially responsible for higher expression of ribosomal proteins and decreased mitochondrial function.ConclusionsOur results suggest that accumulating comorbidities increase levels of innate immune response and lead to DNA damage, senescence-like changes in gene expression and consequently decreased mitochondrial function.

Genome-wide association studies (GWASs) have identified genetic loci associated with the risk of Alzheimer's disease (AD), but the molecular mechanisms by which they confer risk are largely unknown. We conducted a metabolome-wide association study (MWAS) of AD-associated loci from GWASs using untargeted metabolic profiling (metabolomics) by ultraperformance liquid chromatography-mass spectrometry (UPLC-MS). We identified an association of lactosylceramides (LacCer) with AD-related single-nucleotide polymorphisms (SNPs) in

Mutations in hundreds of genes have been associated with formation of human cancer, with different oncogenic lesions prevalent in different cancer types. Yet, the malignant phenotype is simple, characterized by unrestricted growth of cells that invade neighboring healthy tissue and in many cases metastasize to distant organs. One possible hypothesis explaining this dichotomy is that the cancer genes regulate a common set of target genes, which then function as master regulators of essential cancer phenotypes, such as growth, invasion and metastasis. To identify mechanisms that drive the most fundamental feature shared by all tumors – unrestricted cell proliferation – we used a multiomic approach to identify common transcriptional and posttranslational targets of major oncogenic pathways active in different cancer types, and combined this analysis with known regulators of the cell cycle. We identified translation and ribosome biogenesis as common targets of both transcriptional and posttranslational oncogenic pathways. By combining proteomic analysis of clinical samples with functional studies of cell cultures, we also establish NOLC1 as a key node whose convergent regulation both at transcriptional and posttranslational level is critical for tumor cell proliferation. Our results indicate that lineage-specific oncogenic pathways commonly regulate the same set of targets important for growth control, revealing novel key downstream nodes that could be targeted for cancer therapy or chemoprevention.

Funder: Alzheimer's Society; Id: http://dx.doi.org/10.13039/501100000320, Funder: Medical Research Council; Id: http://dx.doi.org/10.13039/501100007155, Alzheimer's disease (AD) is a highly prevalent neurodegenerative disorder. Despite increasing evidence of the importance of metabolic dysregulation in AD, the underlying metabolic changes that may impact amyloid plaque formation are not understood, particularly for late-onset AD. This study analyzed genome-wide association studies (GWAS), transcriptomics, and proteomics data obtained from several data repositories to obtain differentially expressed (DE) multi-omics elements in mouse models of AD. We characterized the metabolic modulation in these data sets using gene ontology, transcription factor, pathway, and cell-type enrichment analyses. A predicted lipid signature was extracted from genome-scale metabolic networks (GSMN) and subsequently validated in a lipidomic data set derived from cortical tissue of ABCA-7 null mice, a mouse model of one of the genes associated with late-onset AD. Moreover, a metabolome-wide association study (MWAS) was performed to further characterize the association between dysregulated lipid metabolism in human blood serum and genes associated with AD risk. We found 203 DE transcripts, 164 DE proteins, and 58 DE GWAS-derived mouse orthologs associated with significantly enriched metabolic biological processes. Lipid and bioenergetic metabolic pathways were significantly over-represented across the AD multi-omics data sets. Microglia and astrocytes were significantly enriched in the lipid-predominant AD-metabolic transcriptome. We also extracted a predicted lipid signature that was validated and robustly modeled class separation in the ABCA7 mice cortical lipidome, with 11 of these lipid species exhibiting statistically significant modulations. MWAS revealed 298 AD single nucleotide polymorphisms-metabolite associations, of which 70% corresponded to lipid classes. These results support the importance of lipid metabolism dysregulation in AD and highlight the suitability of mapping AD multi-omics data into GSMNs to identify metabolic alterations.

Genome-wide association studies (GWASs) have identified genetic loci associated with the risk of Alzheimer’s disease (AD), but the molecular mechanisms by which they confer risk are largely unknown. We conducted a metabolome-wide association study (MWAS) of AD-associated loci from GWASs using untargeted metabolic profiling (metabolomics) by ultraperformance liquid chromatography–mass spectrometry (UPLC-MS). We identified an association of lactosylceramides (LacCer) with AD-related single-nucleotide polymorphisms (SNPs) in ABCA7 ( P = 5.0 × 10 −5 to 1.3 × 10 −44 ). We showed that plasma LacCer concentrations are associated with cognitive performance and genetically modified levels of LacCer are associated with AD risk. We then showed that concentrations of sphingomyelins, ceramides, and hexosylceramides were altered in brain tissue from Abca7 knockout mice, compared with wild type (WT) ( P = 0.049–1.4 × 10 −5 ), but not in a mouse model of amyloidosis. Furthermore, activation of microglia increases intracellular concentrations of hexosylceramides in part through induction in the expression of sphingosine kinase, an enzyme with a high control coefficient for sphingolipid and ceramide synthesis. Our work suggests that the risk for AD arising from functional variations in ABCA7 is mediated at least in part through ceramides. Modulation of their metabolism or downstream signaling may offer new therapeutic opportunities for AD.

On the 24th November 2021 the sequence of a new SARS CoV-2 viral isolate Omicron-B.1.1.529 was announced, containing far more mutations in Spike (S) than previously reported variants. Neutralization titres of Omicron by sera from vaccinees and convalescent subjects infected with early pandemic as well as Alpha, Beta, Gamma, Delta are substantially reduced or fail to neutralize. Titres against Omicron are boosted by third vaccine doses and are high in cases both vaccinated and infected by Delta. Mutations in Omicron knock out or substantially reduce neutralization by most of a large panel of potent monoclonal antibodies and antibodies under commercial development. Omicron S has structural changes from earlier viruses, combining mutations conferring tight binding to ACE2 to unleash evolution driven by immune escape, leading to a large number of mutations in the ACE2 binding site which rebalance receptor affinity to that of early pandemic viruses., A comprehensive analysis of sera from vaccinees, convalescent patients infected previously by multiple variants and potent monoclonal antibodies from early in the COVID-19 pandemic reveals a substantial overall reduction the ability to neutralize the SARS-CoV-2 Omicron variant, which a third vaccine dose seems to ameliorate. Structural analyses of the Omicron RBD suggest a selective pressure enabling the virus bind ACE2 with increased affinity that is offset by other changes in the receptor binding motif that facilitates immune escape.

Funder: Foundation for Liver Research, Funder: Royal Society and a CAMS-UK fellowship, The metabolic syndrome (MetS) is a cluster of cardiovascular risk factors characterised by central obesity, atherogenic dyslipidaemia, and changes in the circulating lipidome; the underlying mechanisms that lead to this lipid remodelling have only been partially elucidated. This study used an integrated "omics" approach (untargeted whole serum lipidomics, targeted proteomics, and lipoprotein lipidomics) to study lipoprotein remodelling and HDL composition in subjects with central obesity diagnosed with MetS (vs. controls). Compared with healthy subjects, MetS patients showed higher free fatty acids, diglycerides, phosphatidylcholines, and triglycerides, particularly those enriched in products of de novo lipogenesis. On the other hand, the "lysophosphatidylcholines to phosphatidylcholines" and "cholesteryl ester to free cholesterol" ratios were reduced, pointing to a lower activity of lecithin cholesterol acyltransferase (LCAT) in MetS; LCAT activity (directly measured and predicted by lipidomic ratios) was positively correlated with high-density lipoprotein cholesterol (HDL-C) and negatively correlated with body mass index (BMI) and insulin resistance. Moreover, many phosphatidylcholines and sphingomyelins were significantly lower in the HDL of MetS patients and strongly correlated with BMI and clinical metabolic parameters. These results suggest that MetS is associated with an impairment of phospholipid metabolism in HDL, partially led by LCAT, and associated with obesity and underlying insulin resistance. This study proposes a candidate strategy to use integrated "omics" approaches to gain mechanistic insights into lipoprotein remodelling, thus deepening the knowledge regarding the molecular basis of the association between MetS and atherosclerosis.

The structure of the metabolic network is highly conserved, but we know little about its evolutionary origins. Key for explaining the early evolution of metabolism is solving a chicken–egg dilemma, which describes that enzymes are made from the very same molecules they produce. The recent discovery of several nonenzymatic reaction sequences that topologically resemble central metabolism has provided experimental support for a “metabolism first” theory, in which at least part of the extant metabolic network emerged on the basis of nonenzymatic reactions. But how could evolution kick-start on the basis of a metal catalyzed reaction sequence, and how could the structure of nonenzymatic reaction sequences be imprinted on the metabolic network to remain conserved for billions of years? We performed an in vitro screening where we add the simplest components of metabolic enzymes, proteinogenic amino acids, to a nonenzymatic, iron-driven reaction network that resembles glycolysis and the pentose phosphate pathway (PPP). We observe that the presence of the amino acids enhanced several of the nonenzymatic reactions. Particular attention was triggered by a reaction that resembles a rate-limiting step in the oxidative PPP. A prebiotically available, proteinogenic amino acid cysteine accelerated the formation of RNA nucleoside precursor ribose-5-phosphate from 6-phosphogluconate. We report that iron and cysteine interact and have additive effects on the reaction rate so that ribose-5-phosphate forms at high specificity under mild, metabolism typical temperature and environmental conditions. We speculate that accelerating effects of amino acids on rate-limiting nonenzymatic reactions could have facilitated a stepwise enzymatization of nonenzymatic reaction sequences, imprinting their structure on the evolving metabolic network., The evolutionary origins of metabolism are largely unknown. This study shows that the prebiotically available proteinogenic amino acid cysteine can promote the metabolism-like rate-limiting formation of ribose-5-phosphate, suggesting that early metabolic pathways could have emerged thought the stepwise enzymatization of non-enzymatic reaction sequences.