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Genetic studies of the metabolome can uncover enzymatic and transport processes shaping human metabolism. Using rare variant aggregation testing based on whole-exome sequencing data to detect genes associated with levels of 1,294 plasma and 1,396 urine metabolites, we discovered 235 gene-metabolite associations, many previously unreported. Complementary approaches (genetic, computational (in silico gene knockouts in whole-body models of human metabolism) and one experimental proof of principle) provided orthogonal evidence that studies of rare, damaging variants in the heterozygous state permit inferences concordant with those from inborn errors of metabolism. Allelic series of functional variants in transporters responsible for transcellular sulfate reabsorption (SLC13A1, SLC26A1) exhibited graded effects on plasma sulfate and human height and pinpointed alleles associated with increased odds of diverse musculoskeletal traits and diseases in the population. This integrative approach can identify new players in incompletely characterized human metabolic reactions and reveal metabolic readouts informative of human traits and diseases., Competing Interests: Competing interests: C.W., S.M., Y.X., R.G. and K.E. are employees of and own shares in Maze Therapeutics. A.K. reports a sponsored research collaboration agreement with Maze Therapeutics. The other authors declare no competing interests., (© 2025. The Author(s).)

Aims: Metformin has been attributed to cardiovascular protection even in the absence of diabetes. Recent observations suggest that metformin influences the gut microbiome. We aimed to investigate the influence of metformin on the gut microbiota and hypertensive target organ damage in hypertensive rats., Methods: Male double transgenic rats overexpressing the human renin and angiotensinogen genes (dTGR), a model of angiotensin II-dependent hypertension, were treated with metformin (300 mg/kg/day) or vehicle from 4 to 7 weeks of age. We assessed gut microbiome composition and function using shotgun metagenomic sequencing and measured blood pressure via radiotelemetry. Cardiac and renal organ damage and inflammation were evaluated by echocardiography, histology, and flow cytometry., Results: Metformin treatment increased the production of short-chain fatty acids (SCFA) acetate and propionate in feces without altering microbial composition and diversity. It significantly reduced systolic and diastolic blood pressure and improved cardiac function, as measured by end-diastolic volume, E/A, and stroke volume despite increased cardiac hypertrophy. Metformin reduced cardiac inflammation by lowering macrophage infiltration and shifting macrophage subpopulations towards a less inflammatory phenotype. The observed improvements in blood pressure, cardiac function, and inflammation correlated with fecal SCFA levels in dTGR. In vitro, acetate and propionate altered M1-like gene expression in macrophages, reinforcing anti-inflammatory effects. Metformin did not affect hypertensive renal damage or microvascular structure., Conclusion: Metformin modulated the gut microbiome, increased SCFA production, and ameliorated blood pressure and cardiac remodeling in dTGR. Our findings confirm the protective effects of metformin in the absence of diabetes, highlighting SCFA as a potential mediators., (© 2024 The Author(s). Acta Physiologica published by John Wiley & Sons Ltd on behalf of Scandinavian Physiological Society.)

Advances in omics technologies have allowed spatially resolved molecular profiling of single cells, providing a window not only into the diversity and distribution of cell types within a tissue, but also into the effects of interactions between cells in shaping the transcriptional landscape. Cells send chemical and mechanical signals which are received by other cells, where they can subsequently initiate context-specific gene regulatory responses. These interactions and their responses shape the individual molecular phenotype of a cell in a given microenvironment. RNAs or proteins measured in individual cells, together with the cells' spatial distribution, provide invaluable information about these mechanisms and the regulation of genes beyond processes occurring independently in each individual cell. "SpaCeNet" is a method designed to elucidate both the intracellular molecular networks (how molecular variables affect each other within the cell) and the intercellular molecular networks (how cells affect molecular variables in their neighbors). This is achieved by estimating conditional independence (CI) relations between captured variables within individual cells and by disentangling these from CI relations between variables of different cells., (© 2024 Schrod et al.; Published by Cold Spring Harbor Laboratory Press.)

Rationale & Objective: Biomarkers that enable better identification of persons with chronic kidney disease (CKD) who are at higher risk for disease progression and adverse events are needed. This study sought to identify urine and plasma metabolites associated with progression of kidney disease., Study Design: Prospective metabolome-wide association study., Setting & Participants: Persons with CKD enrolled in the GCKD (German CKD) study with metabolite measurements, with external validation within the ARIC (Atherosclerosis Risk in Communities) Study., Exposures: 1,513 urine and 1,416 plasma metabolites (Metabolon Inc) measured at study entry using untargeted mass spectrometry., Outcomes: Main end points were kidney failure (KF) and a composite kidney end point (CKE) of KF, estimated glomerular filtration rate<15mL/min/1.73m 2 , or a 40% decrease in estimated glomerular filtration rate. Death from any cause was a secondary end point. After a median of 6.5 years of follow-up, 500 persons had experienced KF, 1,083 had experienced the CKE, and 680 had died., Analytical Approach: Time-to-event analyses using multivariable proportional hazard regression models in a discovery-replication design with external validation., Results: 5,088 GCKD study participants were included in analyses of urine metabolites, and 5,144 were included in analyses of plasma metabolites. Among 182 unique metabolites, 30 were significantly associated with KF, 49 with the CKE, and 163 with death. The strongest association with KF was observed for plasma hydroxyasparagine (HR, 1.95; 95% CI, 1.68-2.25). An unnamed metabolite measured in plasma and urine was significantly associated with KF, the CKE, and death. External validation of the identified associations of metabolites with KF or the CKE revealed directional consistency for 88% of observed associations. Selected associations of 18 metabolites with study outcomes have not been previously reported., Limitations: Use of observational data and semiquantitative metabolite measurements at a single time point., Conclusions: The observed associations between metabolites and KF, the CKE, or death in persons with CKD confirmed previously reported findings and also revealed several associations not previously described. These findings warrant confirmatory research in other study cohorts., Plain-Language Summary: Incomplete understanding of the variability of chronic kidney disease (CKD) progression motivated the search for new biomarkers that would help identify people at increased risk. We explored metabolites in plasma and urine for their association with unfavorable kidney outcomes or death in persons with CKD. Metabolomic analyses revealed 182 metabolites significantly associated with CKD progression or death. Many of these associations confirmed previously reported findings or were validated by analysis in an external study population. Our comprehensive screen of the metabolome serves as a valuable foundation for future investigations into biomarkers associated with CKD progression., (Copyright © 2024 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Aconitate decarboxylase-1 (ACOD1) is expressed by activated macrophages and generates itaconate that exerts anti-microbial and immunoregulatory effects. ACOD1-itaconate is essential for macrophage-mediated control of the intracellular pathogen Coxiella (C.) burnetii , which causes Q fever. Two isomers of itaconate, mesaconate and citraconate, have overlapping yet distinct activity on macrophage metabolism and inflammatory gene expression. Here, we found that all three isomers inhibited the growth of C. burnetii in axenic culture in ACCM-2 medium. However, only itaconate reduced C. burnetii replication efficiently in Acod1 -/- macrophages. In contrast, addition of citraconate strongly increased C. burnetii replication in Acod1 +/- macrophages, whereas mesaconate weakly enhanced bacterial burden in Acod1 -/- macrophages. Analysis of intracellular isomers showed that exogenous citraconate and mesaconate inhibited the generation of itaconate by infected Acod1 +/- macrophages. Uptake of added isomers into Acod1 -/- macrophages was increased after infection for itaconate and mesaconate, but not for citraconate. Mesaconate, but not citraconate, competed with itaconate for uptake into macrophages. Taken together, inhibition of itaconate generation by macrophages and interference with the uptake of extracellular itaconate could be identified as potential mechanisms behind the divergent effects of citraconate and mesaconate on C. burnetii replication in macrophages or in axenic culture., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Siddique, Kellermeier, Ölke, Zhao, Büssow, Oefner, Lührmann, Dettmer and Lang.)

D-2-hydroxyglutarate (D-2-HG) accumulates in patients with acute myeloid leukemia (AML) with mutated isocitrate dehydrogenase (IDH) and in other malignancies. D-2-HG suppresses antitumor T-cell immunity but little is known about potential effects on non-malignant myeloid cells. Here we show that D-2-HG impairs human but not murine dendritic cell differentiation, resulting in a tolerogenic phenotype with low major histocompatibility class II expression. In line with this, IDH-mutated AML blasts exhibited lower expression of HLA-DP and were less susceptible to lysis by HLA-DP-specific T cells. Interestingly, besides its expected impact on DNA demethylation, D-2-HG reprogrammed metabolism towards increased lactate production in dendritic cells and AML. Vitamin C accelerated DNA demethylation, but only the combination of vitamin C and glycolytic inhibition lowered lactate levels and supported major histocompatibility complex class II expression. Our results indicate an unexpected link between the immunosuppressive metabolites 2-HG and lactic acid and suggest a potentially novel therapeutic strategy with combinations of anti-glycolytic drugs and epigenetic modulators (hypomethylating agents) or other therapeutics for the treatment of AML.

Background & Aims: Intratumoral lactate accumulation and acidosis impair T-cell function and antitumor immunity. Interestingly, expression of the lactate transporter monocarboxylate transporter (MCT) 4, but not MCT1, turned out to be prognostic for the survival of patients with rectal cancer, indicating that single MCT4 blockade might be a promising strategy to overcome glycolysis-related therapy resistance., Methods: To determine whether blockade of MCT4 alone is sufficient to improve the efficacy of immune checkpoint blockade (ICB) therapy, we examined the effects of the selective MCT1 inhibitor AZD3965 and a novel MCT4 inhibitor in a colorectal carcinoma (CRC) tumor spheroid model co-cultured with blood leukocytes in vitro and the MC38 murine CRC model in vivo in combination with an antibody against programmed cell death ligand-1(PD-L1)., Results: Inhibition of MCT4 was sufficient to reduce lactate efflux in three-dimensional (3D) CRC spheroids but not in two-dimensional cell-cultures. Co-administration of the MCT4 inhibitor and ICB augmented immune cell infiltration, T-cell function and decreased CRC spheroid viability in a 3D co-culture model of human CRC spheroids with blood leukocytes. Accordingly, combination of MCT4 and ICB increased intratumoral pH, improved leukocyte infiltration and T-cell activation, delayed tumor growth, and prolonged survival in vivo. MCT1 inhibition exerted no further beneficial impact., Conclusions: These findings demonstrate that single MCT4 inhibition represents a novel therapeutic approach to reverse lactic-acid driven immunosuppression and might be suitable to improve ICB efficacy., Competing Interests: Competing interests: The study was conducted in close collaboration with Merck. A.S-H., T.H., and C.H. are employees of Merck. R.F. is an employee of EMD Serono Research & Development Institute, Inc., Billerica, MA, USA, an affiliate of Merck KGaA. S.R. was an employee of EMD Serono Research & Development Institute, Inc., Billerica, MA, USA, an affiliate of Merck KGaA at the time the research was conducted., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Motivation: Mixed molecular data combines continuous and categorical features of the same samples, such as OMICS profiles with genotypes, diagnoses, or patient sex. Like all high-dimensional molecular data, it is prone to incorrect values that can stem from various sources for example the technical limitations of the measurement devices, errors in the sample preparation, or contamination. Most anomaly detection algorithms identify complete samples as outliers or anomalies. However, in most cases, not all measurements of those samples are erroneous but only a few one-dimensional features within the samples are incorrect. These one-dimensional data errors are continuous measurements that are either located outside or inside the normal ranges of their features but in both cases show atypical values given all other continuous and categorical features in the sample. Additionally, categorical anomalies can occur for example when the genotype or diagnosis was submitted wrongly., Results: We introduce ADMIRE (Anomaly Detection using MIxed gRaphical modEls), a novel approach for the detection and correction of anomalies in mixed high-dimensional data. Hereby, we focus on the detection of single (one-dimensional) data errors in the categorical and continuous features of a sample. For that the joint distribution of continuous and categorical features is learned by mixed graphical models, anomalies are detected by the difference between measured and model-based estimations and are corrected using imputation. We evaluated ADMIRE in simulation and by screening for anomalies in one of our own metabolic datasets. In simulation experiments, ADMIRE outperformed the state-of-the-art methods of Local Outlier Factor, stray, and Isolation Forest., Availability and Implementation: All data and code is available at https://github.com/spang-lab/adadmire. ADMIRE is implemented in a Python package called adadmire which can be found at https://pypi.org/project/adadmire., (© The Author(s) 2023. Published by Oxford University Press.)

The beneficial effects of hair follicle stem cells in different animal models of nervous system conditions have been extensively studied. While chick embryo extract (CEE) has been used as a growth medium supplement for these stem cells, this is the first study to show the effect of CEE on them. The rat hair follicle stem cells were isolated and supplemented with 10% fetal bovine serum plus 10% CEE. The migration rate, proliferative capacity and multipotency were evaluated along with morphometric alteration and differentiation direction. The proteome analysis of CEE content identified effective factors of CEE that probably regulate fate and function of stem cells. The CEE enhances the migration rate of stem cells from explanted bulges as well as their proliferation, likely due to activation of AP-1 and translationally controlled tumour protein (TCTP) by thioredoxin found in CEE. The increased length of outgrowth may be the result of cyclic AMP response element binding protein (CREB) phosphorylation triggered by active CamKII contained in CEE. Further, CEE supplementation upregulates the expression of vascular endothelial growth factor (VEGF), brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. The elevated expression of target genes and proteins may be due to CREB, AP-1 and c-Myc activation in these stem cells. Given the increased transcript levels of neurotrophins, VEGF, and the expression of PDGFR-α, S100B, MBP and SOX-10 protein, it is possible that CEE promotes the fate of these stem cells towards Schwann cells., (© 2023 The Authors. Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

The kidneys operate at the interface of plasma and urine by clearing molecular waste products while retaining valuable solutes. Genetic studies of paired plasma and urine metabolomes may identify underlying processes. We conducted genome-wide studies of 1,916 plasma and urine metabolites and detected 1,299 significant associations. Associations with 40% of implicated metabolites would have been missed by studying plasma alone. We detected urine-specific findings that provide information about metabolite reabsorption in the kidney, such as aquaporin (AQP)-7-mediated glycerol transport, and different metabolomic footprints of kidney-expressed proteins in plasma and urine that are consistent with their localization and function, including the transporters NaDC3 (SLC13A3) and ASBT (SLC10A2). Shared genetic determinants of 7,073 metabolite-disease combinations represent a resource to better understand metabolic diseases and revealed connections of dipeptidase 1 with circulating digestive enzymes and with hypertension. Extending genetic studies of the metabolome beyond plasma yields unique insights into processes at the interface of body compartments., (© 2023. The Author(s).)

Glioblastomas are highly malignant brain tumors that derive from brain-tumor-initiating cells (BTICs) and can be subdivided into several molecular subtypes. Metformin is an antidiabetic drug currently under investigation as a potential antineoplastic agent. The effects of metformin on glucose metabolism have been extensively studied, but there are only few data on amino acid metabolism. We investigated the basic amino acid profiles of proneural and mesenchymal BTICs to explore a potential distinct utilization and biosynthesis in these subgroups. We further measured extracellular amino acid concentrations of different BTICs at baseline and after treatment with metformin. Effects of metformin on apoptosis and autophagy were determined using Western Blot, annexin V/7-AAD FACS-analyses and a vector containing the human LC3B gene fused to green fluorescent protein. The effects of metformin on BTICs were challenged in an orthotopic BTIC model. The investigated proneural BTICs showed increased activity of the serine and glycine pathway, whereas mesenchymal BTICs in our study preferably metabolized aspartate and glutamate. Metformin treatment led to increased autophagy and strong inhibition of carbon flux from glucose to amino acids in all subtypes. However, oral treatment with metformin at tolerable doses did not significantly inhibit tumor growth in vivo. In conclusion, we found distinct amino acid profiles of proneural and mesenchymal BTICs, and inhibitory effects of metformin on BTICs in vitro. However, further studies are warranted to better understand potential resistance mechanisms against metformin in vivo.

Background: Coxiella burnetii is a zoonotic pathogen, infecting humans, livestock, pets, birds and ticks. Domestic ruminants such as cattle, sheep, and goats are the main reservoir and major cause of human infection. Infected ruminants are usually asymptomatic, while in humans infection can cause significant disease. Human and bovine macrophages differ in their permissiveness for C. burnetii strains from different host species and of various genotypes and their subsequent host cell response, but the underlying mechanism(s) at the cellular level are unknown., Methods: C. burnetii infected primary human and bovine macrophages under normoxic and hypoxic conditions were analyzed for (i) bacterial replication by CFU counts and immunofluorescence; (ii) immune regulators by westernblot and qRT-PCR; cytokines by ELISA; and metabolites by gas chromatography-mass spectrometry (GC-MS)., Results: Here, we confirmed that peripheral blood-derived human macrophages prevent C. burnetii replication under oxygen-limiting conditions. In contrast, oxygen content had no influence on C. burnetii replication in bovine peripheral blood-derived macrophages. In hypoxic infected bovine macrophages, STAT3 is activated, even though HIF1α is stabilized, which otherwise prevents STAT3 activation in human macrophages. In addition, the TNFα mRNA level is higher in hypoxic than normoxic human macrophages, which correlates with increased secretion of TNFα and control of C. burnetii replication. In contrast, oxygen limitation does not impact TNFα mRNA levels in C. burnetii -infected bovine macrophages and secretion of TNFα is blocked. As TNFα is also involved in the control of C. burnetii replication in bovine macrophages, this cytokine is important for cell autonomous control and its absence is partially responsible for the ability of C. burnetii to replicate in hypoxic bovine macrophages. Further unveiling the molecular basis of macrophage-mediated control of C. burnetii replication might be the first step towards the development of host directed intervention measures to mitigate the health burden of this zoonotic agent., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Mauermeir, Ölke, Hayek, Schulze-Luehrmann, Dettmer, Oefner, Berens, Menge and Lührmann.)

Introduction: Glutamine deficiency is a well-known feature of the tumor environment. Here we analyzed the impact of glutamine deprivation on human myeloid cell survival and function., Methods: Different types of myeloid cells were cultured in the absence or presence of glutamine and/or with L-methionine-S-sulfoximine (MSO), an irreversible glutamine synthetase (GS) inhibitor. GS expression was analyzed on mRNA and protein level. GS activity and the conversion of glutamate to glutamine by myeloid cells was followed by 13C tracing analyses., Results: The absence of extracellular glutamine only slightly affected postmitotic human monocyte to dendritic cell (DC) differentiation, function and survival. Similar results were obtained for monocyte-derived macrophages. In contrast, proliferation of the monocytic leukemia cell line THP-1 was significantly suppressed. While macrophages exhibited high constitutive GS expression, glutamine deprivation induced GS in DC and THP-1. Accordingly, proliferation of THP-1 was rescued by addition of the GS substrate glutamate and 13C tracing analyses revealed conversion of glutamate to glutamine. Supplementation with the GS inhibitor MSO reduced the survival of DC and macrophages and counteracted the proliferation rescue of THP-1 by glutamate., Discussion: Our results show that GS supports myeloid cell survival in a glutamine poor environment. Notably, in addition to suppressing proliferation and survival of tumor cells, the blockade of GS also targets immune cells such as DCs and macrophages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Schoeppe, Babl, Decking, Schönhammer, Siegmund, Bruss, Dettmer, Oefner, Frick, Weigert, Jantsch, Herr, Rehli, Renner and Kreutz.)

Brain-tumor-initiating cells (BTICs) of proneural and mesenchymal origin contribute to the highly malignant phenotype of glioblastoma (GB) and resistance to current therapies. BTICs of different subtypes were challenged with oxidative phosphorylation (OXPHOS) inhibition with metformin to assess the differential effects of metabolic intervention on key resistance features. Whereas mesenchymal BTICs varied according to their invasiveness, they were in general more glycolytic and less responsive to metformin. Proneural BTICs were less invasive, catabolized glucose more via the pentose phosphate pathway, and responded better to metformin. Targeting glycolysis may be a promising approach to inhibit tumor cells of mesenchymal origin, whereas proneural cells are more responsive to OXPHOS inhibition. Future clinical trials exploring metabolic interventions should account for metabolic heterogeneity of brain tumors.

Tachycardiomyopathy is characterised by reversible left ventricular dysfunction, provoked by rapid ventricular rate. While the knowledge of mitochondria advanced in most cardiomyopathies, mitochondrial functions await elucidation in tachycardiomyopathy. Pacemakers were implanted in 61 rabbits. Tachypacing was performed with 330 bpm for 10 days (n = 11, early left ventricular dysfunction) or with up to 380 bpm over 30 days (n = 24, tachycardiomyopathy, TCM). In n = 26, pacemakers remained inactive (SHAM). Left ventricular tissue was subjected to respirometry, metabolomics and acetylomics. Results were assessed for translational relevance using a human-based model: induced pluripotent stem cell derived cardiomyocytes underwent field stimulation for 7 days (TACH-iPSC-CM). TCM animals showed systolic dysfunction compared to SHAM (fractional shortening 37.8 ± 1.0% vs. 21.9 ± 1.2%, SHAM vs. TCM, p < 0.0001). Histology revealed cardiomyocyte hypertrophy (cross-sectional area 393.2 ± 14.5 µm 2 vs. 538.9 ± 23.8 µm 2 , p < 0.001) without fibrosis. Mitochondria were shifted to the intercalated discs and enlarged. Mitochondrial membrane potential remained stable in TCM. The metabolite profiles of ELVD and TCM were characterised by profound depletion of tricarboxylic acid cycle intermediates. Redox balance was shifted towards a more oxidised state (ratio of reduced to oxidised nicotinamide adenine dinucleotide 10.5 ± 2.1 vs. 4.0 ± 0.8, p < 0.01). The mitochondrial acetylome remained largely unchanged. Neither TCM nor TACH-iPSC-CM showed relevantly increased levels of reactive oxygen species. Oxidative phosphorylation capacity of TCM decreased modestly in skinned fibres (168.9 ± 11.2 vs. 124.6 ± 11.45 pmol·O 2 ·s -1 ·mg -1 tissue, p < 0.05), but it did not in isolated mitochondria. The pattern of mitochondrial dysfunctions detected in two models of tachycardiomyopathy diverges from previously published characteristic signs of other heart failure aetiologies., (© 2022. The Author(s).)

Untargeted metabolomics is a promising tool for identifying novel disease biomarkers and unraveling underlying pathomechanisms. Nuclear magnetic resonance (NMR) spectroscopy is particularly suited for large-scale untargeted metabolomics studies due to its high reproducibility and cost effectiveness. Here, one-dimensional (1D) 1 H NMR experiments offer good sensitivity at reasonable measurement times. Their subsequent data analysis requires sophisticated data preprocessing steps, including the extraction of NMR features corresponding to specific metabolites. We developed a novel 1D NMR feature extraction procedure, called Bucket Fuser (BF), which is based on a regularized regression framework with fused group LASSO terms. The performance of the BF procedure was demonstrated using three independent NMR datasets and was benchmarked against existing state-of-the-art NMR feature extraction methods. BF dynamically constructs NMR metabolite features, the widths of which can be adjusted via a regularization parameter. BF consistently improved metabolite signal extraction, as demonstrated by our correlation analyses with absolutely quantified metabolites. It also yielded a higher proportion of statistically significant metabolite features in our differential metabolite analyses. The BF algorithm is computationally efficient and it can deal with small sample sizes. In summary, the Bucket Fuser algorithm, which is available as a supplementary python code, facilitates the fast and dynamic extraction of 1D NMR signals for the improved detection of metabolic biomarkers.

Metabolic fingerprinting by mass spectrometry aims at the comprehensive, semiquantitative analysis of metabolites. Isotope dilution, if successfully implemented, may provide a more reliable, relative quantification. Therefore, the 13 C labeled yeast extract of the IROA TruQuant kit was added as an internal standard (IS) to human urine samples measured in full-scan mode on a high-performance liquid chromatography-time-of-flight mass spectrometer (HPLC-TOFMS) system. The isotope ratio approach enabled the analysis of 112 metabolites. The correlation with reference data did not improve significantly using 12 C/ 13 C ratios compared to absolute 12 C peak areas. Moreover, using an intricate 13 C-labeled standard increased the complexity of the mass spectra, which made correct signal annotation more challenging. On the positive side, the ratio approach helps to reduce batch effects, but it does not perform better than computational methods such as the "removebatcheffect" function in the R package Limma.

Motivation: Estimating the effects of interventions on patient outcome is one of the key aspects of personalized medicine. Their inference is often challenged by the fact that the training data comprises only the outcome for the administered treatment, and not for alternative treatments (the so-called counterfactual outcomes). Several methods were suggested for this scenario based on observational data, i.e. data where the intervention was not applied randomly, for both continuous and binary outcome variables. However, patient outcome is often recorded in terms of time-to-event data, comprising right-censored event times if an event does not occur within the observation period. Albeit their enormous importance, time-to-event data are rarely used for treatment optimization. We suggest an approach named BITES (Balanced Individual Treatment Effect for Survival data), which combines a treatment-specific semi-parametric Cox loss with a treatment-balanced deep neural network; i.e. we regularize differences between treated and non-treated patients using Integral Probability Metrics (IPM)., Results: We show in simulation studies that this approach outperforms the state of the art. Furthermore, we demonstrate in an application to a cohort of breast cancer patients that hormone treatment can be optimized based on six routine parameters. We successfully validated this finding in an independent cohort., Availability and Implementation: We provide BITES as an easy-to-use python implementation including scheduled hyper-parameter optimization (https://github.com/sschrod/BITES). The data underlying this article are available in the CRAN repository at https://rdrr.io/cran/survival/man/gbsg.html and https://rdrr.io/cran/survival/man/rotterdam.html., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press.)

The isocitrate dehydrogenase ( IDH ) mutation status is an indispensable prerequisite for diagnosis of glioma (astrocytoma and oligodendroglioma) according to the WHO classification of brain tumors 2021 and is a potential therapeutic target. Usually, immunohistochemistry followed by sequencing of tumor tissue is performed for this purpose. In clinical routine, however, non-invasive determination of IDH mutation status is desirable in cases where tumor biopsy is not possible and for monitoring neuro-oncological therapies. In a previous publication, we presented reliable prediction of IDH mutation status employing proton magnetic resonance spectroscopy ( 1 H-MRS) on a 3.0 Tesla (T) scanner and machine learning in a prospective cohort of 34 glioma patients. Here, we validated this approach in an independent cohort of 67 patients, for which 1 H-MR spectra were acquired at 1.5 T between 2002 and 2007, using the same data analysis approach. Despite different technical conditions, a sensitivity of 82.6% (95% CI, 61.2-95.1%) and a specificity of 72.7% (95% CI, 57.2-85.0%) could be achieved. We concluded that our 1 H-MRS based approach can be established in a routine clinical setting with affordable effort and time, independent of technical conditions employed. Therefore, the method provides a non-invasive tool for determining IDH status that is well-applicable in an everyday clinical setting.

Accelerated glycolysis leads to secretion and accumulation of lactate and protons in the tumor environment and determines the efficacy of adoptive T cell and checkpoint inhibition therapy. Here, we analyzed effects of lactic acid on different human CD4 T cell subsets and aimed to increase CD4 T cell resistance towards lactic acid. In all CD4 T cell subsets analyzed, lactic acid inhibited metabolic activity (glycolysis and respiration), cytokine secretion, and cell proliferation. Overexpression of the lactate-metabolizing isoenzyme LDHB increased cell respiration and mitigated lactic acid effects on intracellular cytokine production. Strikingly, LDHB-overexpressing cells preferentially migrated into HCT116 tumor spheroids and displayed higher expression of cytotoxic effector molecules. We conclude, that LDHB overexpression might be a promising strategy to increase the efficacy of adoptive T cell transfer therapy.

Uromodulin (UMOD) is a major risk gene for monogenic and complex forms of kidney disease. The encoded kidney-specific protein uromodulin is highly abundant in urine and related to chronic kidney disease, hypertension, and pathogen defense. To gain insights into potential systemic roles, we performed genome-wide screens of circulating uromodulin using complementary antibody-based and aptamer-based assays. We detected 3 and 10 distinct significant loci, respectively. Integration of antibody-based results at the UMOD locus with functional genomics data (RNA-Seq, ATAC-Seq, Hi-C) of primary human kidney tissue highlighted an upstream variant with differential accessibility and transcription in uromodulin-synthesizing kidney cells as underlying the observed cis effect. Shared association patterns with complex traits, including chronic kidney disease and blood pressure, placed the PRKAG2 locus in the same pathway as UMOD. Experimental validation of the third antibody-based locus, B4GALNT2, showed that the p.Cys466Arg variant of the encoded N-acetylgalactosaminyltransferase had a loss-of-function effect leading to higher serum uromodulin levels. Aptamer-based results pointed to enzymes writing glycan marks present on uromodulin and to their receptors in the circulation, suggesting that this assay permits investigating uromodulin's complex glycosylation rather than its quantitative levels. Overall, our study provides insights into circulating uromodulin and its emerging functions.

Local tissue acidosis affects anti-tumor immunity. In contrast, data on tissue pH levels in infected tissues and their impact on antimicrobial activity is sparse. In this study, we assessed the pH levels in cutaneous Leishmania lesions. Leishmania major -infected skin tissue displayed pH levels of 6.7 indicating that lesional pH is acidic. Next, we tested the effect of low extracellular pH on the ability of macrophages to produce leishmanicidal NO and to fight the protozoan parasite Leishmania major . Extracellular acidification led to a marked decrease in both NO production and leishmanicidal activity of lipopolysaccharide (LPS) and interferon γ (IFN-γ)-coactivated macrophages. This was not directly caused by a disruption of NOS2 expression, a shortage of reducing equivalents (NAPDH) or substrate (L-arginine), but by a direct, pH-mediated inhibition of NOS2 enzyme activity. Normalization of intracellular pH significantly increased NO production and antiparasitic activity of macrophages even in an acidic microenvironment. Overall, these findings indicate that low local tissue pH can curtail NO production and leishmanicidal activity of macrophages., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Frick, Hinterland, Renner, Vogl, Babl, Heckscher, Weigert, Weiß, Gläsner, Berger, Oefner, Dettmer, Kreutz, Schatz and Jantsch.)

Due to organ shortage and rising life expectancy the age of organ donors and recipients is increasing. Reliable biomarkers of organ quality that predict successful long-term transplantation outcomes are poorly defined. The aim of this study was the identification of age-related markers of kidney function that might accurately reflect donor organ quality. Histomorphometric, biochemical and molecular parameters were measured in young (3-month-old) and old (24-month-old) male Sprague Dawley rats. In addition to conventional methods, we used urine metabolomics by NMR spectroscopy and gene expression analysis by quantitative RT-PCR to identify markers of ageing relevant to allograft survival. Beside known markers of kidney ageing like albuminuria, changes in the concentration of urine metabolites such as trimethylamine-N-oxide, trigonelline, 2-oxoglutarate, citrate, hippurate, glutamine, acetoacetate, valine and 1-methyl-histidine were identified in association with ageing. In addition, expression of several genes of the toll-like receptor (TLR) pathway, known for their implication in inflammaging, were upregulated in the kidneys of old rats. This study led to the identification of age-related markers of biological allograft age potentially relevant for allograft survival in the future. Among those, urine metabolites and markers of immunity and inflammation, which are highly relevant to immunosuppression in transplant recipients, are promising and deserve further investigation in humans.

Rationale & Objective: Stratification of chronic kidney disease (CKD) patients at risk for progressing to kidney failure requiring kidney replacement therapy (KFRT) is important for clinical decision-making and trial enrollment., Study Design: Four independent prospective observational cohort studies., Setting & Participants: The development cohort comprised 4,915 CKD patients, and 3 independent validation cohorts comprised a total of 3,063. Patients were observed for approximately 5 years., Exposure: 22 demographic, anthropometric, and laboratory variables commonly assessed in CKD patients., Outcome: Progression to KFRT., Analytical Approach: A least absolute shrinkage and selection operator (LASSO) Cox proportional hazards model was fit to select laboratory variables that best identified patients at high risk for KFRT. Model discrimination and calibration were assessed and compared against the 4-variable Tangri (T4) risk equation both in a resampling approach within the development cohort and in the validation cohorts using cause-specific concordance (C) statistics, net reclassification improvement, and calibration graphs., Results: The newly derived 6-variable risk score (Z6) included serum creatinine, albumin, cystatin C, and urea, as well as hemoglobin and the urinary albumin-creatinine ratio. In the the resampling approach, Z6 achieved a median C statistic of 0.909 (95% CI, 0.868-0.937) at 2 years after the baseline visit, whereas the T4 achieved a median C statistic of 0.855 (95% CI, 0.799-0.915). In the 3 independent validation cohorts, the Z6C statistics were 0.894, 0.921, and 0.891, whereas the T4C statistics were 0.882, 0.913, and 0.862., Limitations: The Z6 was both derived and tested only in White European cohorts., Conclusions: A new risk equation based on 6 routinely available laboratory tests facilitates identification of patients with CKD who are at high risk of progressing to KFRT., (Copyright © 2021 National Kidney Foundation, Inc. All rights reserved.)

Background: L-kynurenine is a tryptophan-derived immunosuppressive metabolite and precursor to neurotoxic anthranilate and quinolinate. We evaluated the stereoisomer D-kynurenine as an immunosuppressive therapeutic which is hypothesized to produce less neurotoxic metabolites than L-kynurenine., Methods: L-/D-kynurenine effects on human and murine T cell function were examined in vitro and in vivo (homeostatic proliferation, colitis, cardiac transplant). Kynurenine effects on T cell metabolism were interrogated using [ 13 C] glucose, glutamine and palmitate tracing. Kynurenine was measured in tissues from human and murine tumours and kynurenine-fed mice., Findings: We observed that 1 mM D-kynurenine inhibits T cell proliferation through apoptosis similar to L-kynurenine. Mechanistically, [ 13 C]-tracing revealed that co-stimulated CD4 + T cells exposed to L-/D-kynurenine undergo increased β-oxidation depleting fatty acids. Replenishing oleate/palmitate restored effector T cell viability. We administered dietary D-kynurenine reaching tissue kynurenine concentrations of 19 μM, which is close to human kidney (6 μM) and head and neck cancer (14 μM) but well below the 1 mM required for apoptosis. D-kynurenine protected Rag1 -/- mice from autoimmune colitis in an aryl-hydrocarbon receptor dependent manner but did not attenuate more stringent immunological challenges such as antigen mismatched cardiac allograft rejection., Interpretation: Our dietary kynurenine model achieved tissue concentrations at or above human cancer kynurenine and exhibited only limited immunosuppression. Sub-suppressive kynurenine concentrations in human cancers may limit the responsiveness to indoleamine 2,3-dioxygenase inhibition evaluated in clinical trials., Funding: The study was supported by the NIH, the Else Kröner-Fresenius-Foundation, Laffey McHugh foundation, and American Society of Nephrology., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Rationale & Objective: Mechanisms underlying the variable course of disease progression in patients with chronic kidney disease (CKD) are incompletely understood. The aim of this study was to identify novel biomarkers of adverse kidney outcomes and overall mortality, which may offer insights into pathophysiologic mechanisms., Study Design: Metabolome-wide association study., Setting & Participants: 5,087 patients with CKD enrolled in the observational German Chronic Kidney Disease Study., Exposures: Measurements of 1,487 metabolites in urine., Outcomes: End points of interest were time to kidney failure (KF), a combined end point of KF and acute kidney injury (KF+AKI), and overall mortality., Analytical Approach: Statistical analysis was based on a discovery-replication design (ratio 2:1) and multivariable-adjusted Cox regression models., Results: After a median follow-up of 4 years, 362 patients died, 241 experienced KF, and 382 experienced KF+AKI. Overall, we identified 55 urine metabolites whose levels were significantly associated with adverse kidney outcomes and/or mortality. Higher levels of C-glycosyltryptophan were consistently associated with all 3 main end points (hazard ratios of 1.43 [95% CI, 1.27-1.61] for KF, 1.40 [95% CI, 1.27-1.55] for KF+AKI, and 1.47 [95% CI, 1.33-1.63] for death). Metabolites belonging to the phosphatidylcholine pathway showed significant enrichment. Members of this pathway contributed to the improvement of the prediction performance for KF observed when multiple metabolites were added to the well-established Kidney Failure Risk Equation., Limitations: Findings among patients of European ancestry with CKD may not be generalizable to the general population., Conclusions: Our comprehensive screen of the association between urine metabolite levels and adverse kidney outcomes and mortality identifies metabolites that predict KF and represents a valuable resource for future studies of biomarkers of CKD progression., (Copyright © 2021 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

Background: Polypharmacy is common among patients with CKD, but little is known about the urinary excretion of many drugs and their metabolites among patients with CKD., Methods: To evaluate self-reported medication use in relation to urine drug metabolite levels in a large cohort of patients with CKD, the German Chronic Kidney Disease study, we ascertained self-reported use of 158 substances and 41 medication groups, and coded active ingredients according to the Anatomical Therapeutic Chemical Classification System. We used a nontargeted mass spectrometry-based approach to quantify metabolites in urine; calculated specificity, sensitivity, and accuracy of medication use and corresponding metabolite measurements; and used multivariable regression models to evaluate associations and prescription patterns., Results: Among 4885 participants, there were 108 medication-drug metabolite pairs on the basis of reported medication use and 78 drug metabolites. Accuracy was excellent for measurements of 36 individual substances in which the unchanged drug was measured in urine (median, 98.5%; range, 61.1%-100%). For 66 pairs of substances and their related drug metabolites, median measurement-based specificity and sensitivity were 99.2% (range, 84.0%-100%) and 71.7% (range, 1.2%-100%), respectively. Commonly prescribed medications for hypertension and cardiovascular risk reduction-including angiotensin II receptor blockers, calcium channel blockers, and metoprolol-showed high sensitivity and specificity. Although self-reported use of prescribed analgesics (acetaminophen, ibuprofen) was <3% each, drug metabolite levels indicated higher usage (acetaminophen, 10%-26%; ibuprofen, 10%-18%)., Conclusions: This comprehensive screen of associations between urine drug metabolite levels and self-reported medication use supports the use of pharmacometabolomics to assess medication adherence and prescription patterns in persons with CKD, and indicates under-reported use of medications available over the counter, such as analgesics., (Copyright © 2021 by the American Society of Nephrology.)

Background: Gastrointestinal dysfunction is a frequent and disabling manifestation of autoimmune polyendocrine syndrome type 1 (APS-1), a rare monogenic multiorgan autoimmune disease caused by the loss of central AIRE-controlled immune tolerance., Objectives: This study aimed to understand the role of the gut microbiome in APS-1 symptoms and potentially alleviate common gastrointestinal symptoms by probiotic intervention., Methods: This study characterized the fecal microbiomes of 28 patients with APS-1 and searched for associations with gastrointestinal symptoms, circulating anti-cytokine autoantibodies, and tryptophan-related metabolites. Additionally, daily doses of the probiotic Lactobacillus rhamnosus GG were administered for 3 months., Results: Of 581 metagenomic operational taxonomic units (mOTUs) characterized in total, 14 were significantly associated with patients with APS-1 compared with healthy controls, with 6 mOTUs depleted and 8 enriched in patients with APS-1. Four overabundant mOTUs were significantly associated with severity of constipation. Phylogenetically conserved microbial associations with autoantibodies against cytokines were observed. After the 3-month intervention with the probiotic L rhamnosus GG, a subset of gastrointestinal symptoms were alleviated. L rhamnosus GG abundance was increased postintervention and corresponded with decreased abundances of Alistipes onderdonkii and Collinsella aerofaciens, 2 species positively associated with severity of diarrhea in patients with APS-1., Conclusions: The APS-1 microbiome correlates with several APS-1 symptoms, some of which are alleviated after a 3-month L rhamnosus GG intervention. Autoantibodies against cytokines appear to shape the gut microbiome by positively correlating with a taxonomically consistent group of bacteria., (Copyright © 2021. Published by Elsevier Inc.)

NMR spectroscopy is a widely used method for the detection and quantification of metabolites in complex biological fluids. However, the large number of metabolites present in a biological sample such as urine or plasma leads to considerable signal overlap in one-dimensional NMR spectra, which in turn hampers both signal identification and quantification. As a consequence, we have developed an easy to use R -package that allows the fully automated deconvolution of overlapping signals in the underlying Lorentzian line-shapes. We show that precise integral values are computed, which are required to obtain both relative and absolute quantitative information. The algorithm is independent of any knowledge of the corresponding metabolites, which also allows the quantitative description of features of yet unknown identity.

Purpose: 3-indoxyl sulfate (3-IS) is an indole metabolism byproduct produced by commensal gut bacteria and excreted in the urine; low urinary 3-IS has been associated with increased mortality in bone marrow transplant recipients. This study investigated urinary 3-IS and patient outcomes in the ICU., Materials and Methods: Prospective study that collected urine samples, rectal swabs, and clinical data on 78 adult ICU patients at admission and again 72 h later. Urine was analyzed for 3-IS by mass spectrometry., Results: Median urinary 3-IS levels were 17.1 μmol/mmol creatinine (IQR 9.5 to 26.2) at admission and 15.6 (IQR 4.2 to 30.7) 72 h later. 22% of patients had low 3-IS (≤6.9 μmol/mmol) on ICU admission and 28% after 72 h. Low 3-IS at 72 h was associated with fewer ICU-free days (22.5 low versus 26 high, p = 0.03) and with death during one year of follow-up (36% low versus 9% high 3-IS, p < 0.01); there was no detectable difference in 30-day mortality (18% low versus 5% high, p = 0.07)., Conclusions: Low urinary 3-IS level 72 h after ICU admission was associated with fewer ICU-free days and with increased one-year but not 30-day mortality. Further studies should investigate urinary 3-IS as an ICU biomarker., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Knowledge about converging disease mechanisms in the heterogeneous syndrome amyotrophic lateral sclerosis (ALS) is rare, but may lead to therapies effective in most ALS cases. Previously, we identified serum microRNAs downregulated in familial ALS, the majority of sporadic ALS patients, but also in presymptomatic mutation carriers. A 5-nucleotide sequence motif (GDCGG; D = G, A or U) was strongly enriched in these ALS-related microRNAs. We hypothesized that deregulation of protein(s) binding predominantly to this consensus motif was responsible for the ALS-linked microRNA fingerprint. Using microRNA pull-down assays combined with mass spectrometry followed by extensive biochemical validation, all members of the fragile X protein family, FMR1, FXR1 and FXR2, were identified to directly and predominantly interact with GDCGG microRNAs through their structurally disordered RGG/RG domains. Preferential association of this protein family with ALS-related microRNAs was confirmed by in vitro binding studies on a transcriptome-wide scale. Immunohistochemistry of lumbar spinal cord revealed aberrant expression level and aggregation of FXR1 and FXR2 in C9orf72- and FUS-linked familial ALS, but also patients with sporadic ALS. Further analysis of ALS autopsies and induced pluripotent stem cell-derived motor neurons with FUS mutations showed co-aggregation of FXR1 with FUS. Hence, our translational approach was able to take advantage of blood microRNAs to reveal CNS pathology, and suggests an involvement of the fragile X-related proteins in familial and sporadic ALS already at a presymptomatic stage. The findings may uncover disease mechanisms relevant to many patients with ALS. They furthermore underscore the systemic, extra-CNS aspect of ALS., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain.)

In recent years, onco-metabolites like D-2-hydroxyglutarate, which is produced in isocitrate dehydrogenase-mutated tumors, have gained increasing interest. Here, we report a metabolite in human specimens that is closely related to 2-hydroxyglutarate: the intramolecular ester of 2-hydroxyglutarate, 2-hydroxyglutarate-γ-lactone. Using 13 C 5 -L-glutamine tracer analysis, we showed that 2-hydroxyglutarate is the endogenous precursor of 2-hydroxyglutarate-lactone and that there is a high exchange between these two metabolites. Lactone formation does not depend on mutated isocitrate dehydrogenase, but its formation is most probably linked to transport processes across the cell membrane and favored at low environmental pH. Furthermore, human macrophages showed not only striking differences in uptake of 2-hydroxyglutarate and its lactone but also in the enantiospecific hydrolysis of the latter. Consequently, 2-hydroxyglutarate-lactone may play a critical role in the modulation of the tumor microenvironment.

Mitochondria are important regulators of macrophage polarisation. Here, we show that arginase-2 (Arg2) is a microRNA-155 (miR-155) and interleukin-10 (IL-10) regulated protein localized at the mitochondria in inflammatory macrophages, and is critical for IL-10-induced modulation of mitochondrial dynamics and oxidative respiration. Mechanistically, the catalytic activity and presence of Arg2 at the mitochondria is crucial for oxidative phosphorylation. We further show that Arg2 mediates this process by increasing the activity of complex II (succinate dehydrogenase). Moreover, Arg2 is essential for IL-10-mediated downregulation of the inflammatory mediators succinate, hypoxia inducible factor 1α (HIF-1α) and IL-1β in vitro. Accordingly, HIF-1α and IL-1β are highly expressed in an LPS-induced in vivo model of acute inflammation using Arg2 -/- mice. These findings shed light on a new arm of IL-10-mediated metabolic regulation, working to resolve the inflammatory status of the cell.

Metabolite levels in urine may provide insights into genetic mechanisms shaping their related pathways. We therefore investigate the cumulative contribution of rare, exonic genetic variants on urine levels of 1487 metabolites and 53,714 metabolite ratios among 4864 GCKD study participants. Here we report the detection of 128 significant associations involving 30 unique genes, 16 of which are known to underlie inborn errors of metabolism. The 30 genes are strongly enriched for shared expression in liver and kidney (odds ratio = 65, p-FDR = 3e-7), with hepatocytes and proximal tubule cells as driving cell types. Use of UK Biobank whole-exome sequencing data links genes to diseases connected to the identified metabolites. In silico constraint-based modeling of gene knockouts in a virtual whole-body, organ-resolved metabolic human correctly predicts the observed direction of metabolite changes, highlighting the potential of linking population genetics to modeling. Our study implicates candidate variants and genes for inborn errors of metabolism.

Cellular metabolism is dynamically regulated in NK cells and strongly influences their responses. Metabolic dysfunction is linked to defective NK cell responses in diseases such as obesity and cancer. The transcription factors, sterol regulatory element binding protein (SREBP) and cMyc, are crucial for controlling NK cell metabolic and functional responses, though the mechanisms involved are not fully understood. This study reveals a new role for SREBP in NK cells in supporting de novo polyamine synthesis through facilitating elevated cMyc expression. Polyamines have diverse roles and their de novo synthesis is required for NK cell glycolytic and oxidative metabolism and to support optimal NK cell effector functions. When NK cells with impaired SREBP activity were supplemented with exogenous polyamines, NK cell metabolic defects were not rescued but these NK cells displayed significant improvement in some effector functions. One role for polyamines is in the control of protein translation where spermidine supports the posttranslational hypusination of translation factor eIF5a. Pharmacological inhibition of hypusination also impacts upon NK cell metabolism and effector function. Considering recent evidence that cholesterol-rich tumor microenvironments inhibit SREBP activation and drive lymphocyte dysfunction, this study provides key mechanistic insight into this tumor-evasion strategy., (© 2020 Wiley-VCH GmbH.)

Background: Lipofilling is one of the most often performed surgical procedures in plastic and reconstructive surgery. Lipoaspirates provide a ready source of stem cells and secreted factors that contribute to neoangiogenesis and fat graft survival. However, the regulations about the enrichment of these beneficial cells and factors are ambiguous. In this study, the authors tested whether a combination of centrifugation and homogenization allowed the enrichment of viable stem cells in lipoaspirates through the selective removal of tumescent solution, blood, and released lipids without significantly affecting the cell secretome., Methods: Human lipoaspirate was harvested from six different patients using water jet-assisted liposuction. Lipoaspirate was homogenized by first centrifugation (3584 rpm for 2 minutes), shear strain (10 times intersyringe processing), and second centrifugation (3584 rpm for 2 minutes). Stem cell enrichment was shown by cell counting after stem cell isolation. Lipoaspirate from different processing steps (unprocessed, after first centrifugation, after homogenization, after second centrifugation) was incubated in serum-free cell culture medium for mass spectrometric analysis of secreted proteins., Results: Lipoaspirate homogenization leads to a significant 2.6 ± 1.75-fold enrichment attributable to volume reduction without reducing the viability of the stem cells. Protein composition of the secretome did not change significantly after tissue homogenization. Considering the enrichment effects, there were no significant differences in the protein concentration of the 83 proteins found in all processing steps., Conclusions: Stem cells can be enriched mechanically without significantly affecting the composition of secreted proteins. Shear-assisted enrichment of lipoaspirate constitutes no substantial manipulation of the cells' secretome.

Isocitrate dehydrogenase ( IDH)-1 mutation is an important prognostic factor and a potential therapeutic target in glioma. Immunohistological and molecular diagnosis of IDH mutation status is invasive. To avoid tumor biopsy, dedicated spectroscopic techniques have been proposed to detect D-2-hydroxyglutarate (2-HG), the main metabolite of IDH , directly in vivo. However, these methods are technically challenging and not broadly available. Therefore, we explored the use of machine learning for the non-invasive, inexpensive and fast diagnosis of IDH status in standard 1 H-magnetic resonance spectroscopy ( 1 H-MRS). To this end, 30 of 34 consecutive patients with known or suspected glioma WHO grade II-IV were subjected to metabolic positron emission tomography (PET) imaging with O-(2- 18 F-fluoroethyl)-L-tyrosine ( 18 F-FET) for optimized voxel placement in 1 H-MRS. Routine 1 H-magnetic resonance ( 1 H-MR) spectra of tumor and contralateral healthy brain regions were acquired on a 3 Tesla magnetic resonance (3T-MR) scanner, prior to surgical tumor resection and molecular analysis of IDH status. Since 2-HG spectral signals were too overlapped for reliable discrimination of IDH mutated ( IDHmut ) and IDH wild-type ( IDHwt ) glioma, we used a nested cross-validation approach, whereby we trained a linear support vector machine (SVM) on the complete spectral information of the 1 H-MRS data to predict IDH status. Using this approach, we predicted IDH status with an accuracy of 88.2%, a sensitivity of 95.5% (95% CI, 77.2-99.9%) and a specificity of 75.0% (95% CI, 42.9-94.5%), respectively. The area under the curve (AUC) amounted to 0.83. Subsequent ex vivo 1 H-nuclear magnetic resonance ( 1 H-NMR) measurements performed on metabolite extracts of resected tumor material (eight specimens) revealed myo-inositol (M-ins) and glycine (Gly) to be the major discriminators of IDH status. We conclude that our approach allows a reliable, non-invasive, fast and cost-effective prediction of IDH status in a standard clinical setting.

The spectral resolution of 2D 1H-13C heteronuclear single quantum coherence (1H-13C-HSQC) nuclear magnetic resonance (NMR) spectra facilitates both metabolite identification and quantification in nuclear magnetic resonance-based metabolomics. However, quantification is complicated by variations in magnetization transfer, which among others originate mainly from scalar coupling differences. Methods that compensate for variation in scalar coupling include the generation of calibration factors for individual signals or the use of additional pulse sequence schemes such as quantitative HSQC (Q-HSQC) that suppress the J CH -dependence by modulating the polarization transfer delays of HSQC or, additionally, employ a pure-shift homodecoupling approach in the 1H dimension, such as Quantitative, Perfected and Pure Shifted HSQC (QUIPU-HSQC). To test the quantitative accuracy of these three methods, employing a 600 MHz NMR spectrometer equipped with a helium cooled cryoprobe, a Latin-square design that covered the physiological concentration ranges of 10 metabolites was used. The results show the suitability of all three methods for the quantification of highly abundant metabolites. However, the substantially increased residual water signal observed in QUIPU-HSQC spectra impeded the quantification of low abundant metabolites located near the residual water signal, thus limiting its utility in high-throughput metabolite fingerprinting studies.