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Next to the use of 1D and 2D-gel purification systems, proteome studies can also be made starting from total digests. The resulting peptide mixture is generally too complex in order to purify and to analyze each individual peptide, leading to a shotgun approach. In order to solve this problem, we have recently developed a chromatographic sorting system able to select a subset of peptides characterized by rare amino acids or functional groups. These sorted peptides represent their corresponding parent proteins present in the original mixture. The sorting procedure is based on the concept of diagonal chromatography, consisting of two identical chromatographic separations with a specific chemical or enzymatic alteration, introduced in between the runs on a subset of peptides. Altered peptides will shift in the second chromatographic run compared to their position in the primary run and therefore separate from the bulk of non-altered peptides. This concept was adapted in order to handle highly complex mixtures such as tryptic digests of total cell lysates and was named COmbined FRActional DIagonal Chromatographic (COFRADIC[sup TM] ). The procedure is illustrated by the sorting and MS-MS-based identification of methionine-containing peptides from an E. coli and human platelet proteome. Results of a full cysteine-peptide proteome of human platelets are also shown. COFRADIC[sup TM] also allows specific selection of the N-terminal peptides of proteins present in a platelet lysate. This strategy reduces the complexity to a one peptide – one protein ratio, leading to a much better protein coverage. In addition, it allows to study protein processing in a global context or to select for proteins with an in vivo α -N-acetyl group. The technology can be extended to any amino acid side chain or post-translational modification, which can be fully automated and leads to the discovery of a broad range of proteins. [ABSTRACT FROM AUTHOR]

Endothelial cells (ECs) not only form passive blood conduits but actively contribute to nutrient transport and organ homeostasis. The role of ECs in glucose homeostasis is, however, poorly understood. Here, we show that, in skeletal muscle, endothelial glucose transporter 1 (Glut1/Slc2a1) controls glucose uptake via vascular metabolic control of muscle-resident macrophages without affecting transendothelial glucose transport. Lowering endothelial Glut1 via genetic depletion (Glut1 ΔEC ) or upon a short-term high-fat diet increased angiocrine osteopontin (OPN/Spp1) secretion. This promoted resident muscle macrophage activation and proliferation, which impaired muscle insulin sensitivity. Consequently, co-deleting Spp1 from ECs prevented macrophage accumulation and improved insulin sensitivity in Glut1 ΔEC mice. Mechanistically, Glut1-dependent endothelial glucose metabolic rewiring increased OPN in a serine metabolism-dependent fashion. Our data illustrate how the glycolytic endothelium creates a microenvironment that controls resident muscle macrophage phenotype and function and directly links resident muscle macrophages to the maintenance of muscle glucose homeostasis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: In lung transplantation (LuTx), various ischemic phases exist, yet the rewarming ischemia time (RIT) during implantation has often been overlooked. During RIT, lungs are deflated and exposed to the body temperature in the recipient's chest cavity. Our prior clinical findings demonstrated that prolonged RIT increases the risk of primary graft dysfunction. However, the molecular mechanisms of rewarming ischemic injury in this context remain unexplored. We aimed to characterize the rewarming ischemia phase during LuTx by measuring organ temperature and comparing transcriptome and metabolome profiles in tissue obtained at the end versus the start of implantation., Methods: In a clinical observational study, 34 double-LuTx with ice preservation were analyzed. Lung core and surface temperature (n=65 and 55 lungs) was measured during implantation. Biopsies (n=59 lungs) were wedged from right middle lobe and left lingula at start and end of implantation. Tissue transcriptomic and metabolomic profiling were performed., Results: Temperature increased rapidly during implantation, reaching core/surface temperatures of 21.5°C/25.4°C within 30min. Transcriptomics showed increased pro-inflammatory signaling and oxidative stress at the end of implantation. Upregulation of NLRP3 and NFKB1 correlated with RIT. Metabolomics indicated elevated levels of amino acids, hypoxanthine, uric acid, cysteineglutathione disulfide alongside decreased levels of glucose and carnitines. Arginine, tyrosine, and 1-carboxyethylleucine showed correlation with incremental RIT., Conclusions: The final rewarming ischemia phase in LuTx involves rapid organ rewarming, accompanied by transcriptomic and metabolomic changes indicating pro-inflammatory signaling and disturbed cell metabolism. Limiting implantation time and lung cooling represent potential interventions to alleviate rewarming ischemic injury., (Copyright © 2024 International Society for the Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Chronic stress has been shown to induce hyperglycemia in both peripheral blood and the brain, yet the detailed mechanisms of glucose metabolism under stress remain unclear. Utilizing 13 C 6 -labeled glucose to trace metabolic pathways, our study investigated the impact of stress by chronic social defeat (CSD) on glucose metabolites in the liver and brain one week post-stress. We observed a reduction in 13 C 6 -enrichment of glucose metabolites in the liver, contrasting with unchanged levels in the brain. Notably, hepatic glycogen levels were reduced while lactate concentrations were elevated, suggesting lactate as an alternative energy source during stress. Long-term effects were also examined, revealing normalized blood glucose levels and restored glycogen stores in the liver three weeks post-CSD, despite sustained increases in food intake. This normalization is hypothesized to result from diminished glucagon levels leading to reduced glycogen phosphorylase activity. Our findings highlight a temporal shift in glucose metabolism, with hyperglycemia and glycogen depletion in the liver early after CSD, followed by a later phase of metabolic stabilization. These results underscore the liver's critical role in adapting to CSD and provide insights into the metabolic adjustments that maintain glucose homeostasis under prolonged stress conditions., (© 2024. The Author(s).)

Aims: During embryonic development, arteriovenous (AV) differentiation ensures proper blood vessel formation and maturation. Defects in arterial or venous identity cause inappropriate fusion of vessels, resulting in atypical shunts, so-called arteriovenous malformations (AVM). Currently, the mechanism behind AVM formation remains unclear and treatment options are fairly limited. Mammalian AV differentiation is initiated before the onset of blood flow in the embryo; however, this pre-flow mechanism is poorly understood. Here, we aimed to unravel the role of Smad1/5 signalling in pre-flow arterial identity, and in the process uncovered an unexpected control mechanism of Smad1/5 signalling., Methods and Results: We establish that despite Notch1 being expressed in the pre-flow mouse embryo, it is not activated, nor is it necessary for the expression of the earliest arterial genes in the dorsal aortae (i.e., Hey1 and Gja4). Furthermore, interrupting blood flow by using the Ncx1 KO model completely prevents the activation of Notch1 signalling, suggesting a strong role of shear stress in maintaining arterial identity. We demonstrate that early expression of Hey1 and Gja4 requires SMAD1/5 signalling. Using embryo cultures, we show that Smad1/5 signalling is activated through the Alk1/Alk5/TGFβR2 receptor complex, with TGFβ1 as a necessary ligand. Furthermore, our findings demonstrate that early arterial gene expression requires the acetylation of Smad1/5 proteins, rendering them more sensitive to TGFβ1 stimulation. Blocking acetyl-CoA production prevents pre-flow arterial expression of Hey1 and Gja4, while stabilizing acetylation rescues their expression., Conclusions: Our findings highlight the importance of the acetyl-CoA production in the cell and provide a novel control mechanism of Smad1/5 signalling involving protein acetylation. As disturbed canonical Smad1/5 signalling is involved in several vascular conditions, our results offer new insights in treatment options for circumventing canonical Smad1/5 signalling., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Despite the implementation of personalized medicine, patients with metastatic CRC (mCRC) still have a dismal overall survival due to the frequent occurrence of acquired resistance mechanisms thereby leading to clinical relapse. Understanding molecular mechanisms that support acquired resistance to anti-EGFR targeted therapy in mCRC is therefore clinically relevant and key to improving patient outcomes. Here, we observe distinct metabolic changes between cetuximab-resistant CRC cell populations, with in particular an increased glycolytic activity in KRAS-mutant cetuximab-resistant CRC cells (LIM1215 and OXCO2) but not in KRAS-amplified resistant DiFi cells. We show that cetuximab-resistant LIM1215 and OXCO2 cells have the capacity to recycle glycolysis-derived lactate to sustain their growth capacity. This is associated with an upregulation of the lactate importer MCT1 at both transcript and protein levels. Pharmacological inhibition of MCT1, with AR-C155858, reduces the uptake and oxidation of lactate and impairs growth capacity in cetuximab-resistant LIM1215 cells both in vitro and in vivo. This study identifies MCT1-dependent lactate utilization as a clinically actionable, metabolic vulnerability to overcome KRAS-mutant-mediated acquired resistance to anti-EGFR therapy in CRC., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: S.A. reports personal fees from MSD Italia and a patent (international PCT patent application No. WO 2023/199255 and Italian patent application No. 102022000007535) outside the submitted work. A.Ba. reports receipt of grants/research support from Neophore, AstraZeneca and Boehringer Ingelheim and honoraria/consultation fees from Guardant Health and Inivata. A.Ba. is a stock shareholder of Neophore and Kither Biotech. A.Ba. is an advisory board member for Inivata, Neophore, Roche/Genentech. The other authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Many individuals with cancer are resistant to immunotherapies. Here, we identify the gene encoding the pyrimidine salvage pathway enzyme cytidine deaminase (CDA) among the top upregulated metabolic genes in several immunotherapy-resistant tumors. We show that CDA in cancer cells contributes to the uridine diphosphate (UDP) pool. Extracellular UDP hijacks immunosuppressive tumor-associated macrophages (TAMs) through its receptor P2Y 6 . Pharmacologic or genetic inhibition of CDA in cancer cells (or P2Y 6 in TAMs) disrupts TAM-mediated immunosuppression, promoting cytotoxic T cell entry and susceptibility to anti-programmed cell death protein 1 (anti-PD-1) treatment in resistant pancreatic ductal adenocarcinoma (PDAC) and melanoma models. Conversely, CDA overexpression in CDA-depleted PDACs or anti-PD-1-responsive colorectal tumors or systemic UDP administration (re)establishes resistance. In individuals with PDAC, high CDA levels in cancer cells correlate with increased TAMs, lower cytotoxic T cells and possibly anti-PD-1 resistance. In a pan-cancer single-cell atlas, CDA high cancer cells match with T cell cytotoxicity dysfunction and P2RY6 high TAMs. Overall, we suggest CDA and P2Y 6 as potential targets for cancer immunotherapy., (© 2024. The Author(s).)

Solid tumors are highly reliant on lipids for energy, growth, and survival. In prostate cancer, the activity of the androgen receptor (AR) is associated with reprogramming of lipid metabolic processes. Here, we identified acyl-CoA synthetase medium chain family members 1 and 3 (ACSM1 and ACSM3) as AR-regulated mediators of prostate cancer metabolism and growth. ACSM1 and ACSM3 were upregulated in prostate tumors compared with nonmalignant tissues and other cancer types. Both enzymes enhanced proliferation and protected prostate cancer cells from death in vitro, whereas silencing ACSM3 led to reduced tumor growth in an orthotopic xenograft model. ACSM1 and ACSM3 were major regulators of the prostate cancer lipidome and enhanced energy production via fatty acid oxidation. Metabolic dysregulation caused by loss of ACSM1/3 led to mitochondrial oxidative stress, lipid peroxidation, and cell death by ferroptosis. Conversely, elevated ACSM1/3 activity enabled prostate cancer cells to survive toxic levels of medium chain fatty acids and promoted resistance to ferroptosis-inducing drugs and AR antagonists. Collectively, this study reveals a tumor-promoting function of medium chain acyl-CoA synthetases and positions ACSM1 and ACSM3 as key players in prostate cancer progression and therapy resistance. Significance: Androgen receptor-induced ACSM1 and ACSM3 mediate a metabolic pathway in prostate cancer that enables the utilization of medium chain fatty acids for energy production, blocks ferroptosis, and drives resistance to clinically approved antiandrogens., (©2024 American Association for Cancer Research.)

Objective: To investigate whether ischemia alters donor kidney metabolism and whether these changes are associated with organ function., Background: An unmet need in kidney transplantation is the ability to predict posttransplant organ function before transplantation. Key to such viability testing is a profound understanding of the organ's complex biochemistry and how ischemia, inevitable during the transplantation process, influences this., Methods: First, metabolic changes in perfusate glucose, lactate, and 20 amino acids, induced by no, 1 hour of warm, or 22 hours of cold ischemia, were investigated during 4-hour perfusion of pig kidneys with autologous whole blood (n = 6/group), simulating the ischemia-reperfusion phase of transplantation. Next, we confirmed similar metabolic changes during normothermic preservation of pigs (n = 3/group; n = 4 for cold ischemia) and discarded human kidneys (n = 6) perfused with a red blood cell-based perfusate., Results: At 2 hours of perfusion with autologous whole blood, abundances of 17/20 amino acids were significantly different between groups, reflecting the type of ischemia. Amino acid changes at 15 minutes and 2 hours of perfusion correlated with future kidney function during perfusion. Similar metabolic patterns were observed during perfusion preservation of pig and discarded human donor kidneys, suggesting an opportunity to assess kidney viability before transplantation., Conclusions: Perfusate metabolite changes during normothermic kidney perfusion represent a unique noninvasive opportunity to assess graft viability. These findings now need validation in transplant studies., Competing Interests: B.G. and I.J. are listed as coinventors of a patent application on methods and applications of analyzing the perfusate of an ex situ perfused kidney (PCT/EP2023/052628). I.J. received speaker fees from XVIVO perfusion paid to her institution. The remaining authors report no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Endothelial cells (ECs) are highly glycolytic, but whether they generate glycolytic intermediates via gluconeogenesis (GNG) in glucose-deprived conditions remains unknown. Here, we report that glucose-deprived ECs upregulate the GNG enzyme PCK2 and rely on a PCK2-dependent truncated GNG, whereby lactate and glutamine are used for the synthesis of lower glycolytic intermediates that enter the serine and glycerophospholipid biosynthesis pathways, which can play key roles in redox homeostasis and phospholipid synthesis, respectively. Unexpectedly, however, even in normal glucose conditions, and independent of its enzymatic activity, PCK2 silencing perturbs proteostasis, beyond its traditional GNG role. Indeed, PCK2-silenced ECs have an impaired unfolded protein response, leading to accumulation of misfolded proteins, which due to defective proteasomes and impaired autophagy, results in the accumulation of protein aggregates in lysosomes and EC demise. Ultimately, loss of PCK2 in ECs impaired vessel sprouting. This study identifies a role for PCK2 in proteostasis beyond GNG., (© 2024. The Author(s).)

Background and Aims: Acute-on-chronic liver failure (ACLF) is a complication of cirrhosis characterized by multiple organ failure and high short-term mortality. The pathophysiology of ACLF involves elevated systemic inflammation leading to organ failure, along with immune dysfunction that heightens susceptibility to bacterial infections. However, it is unclear how these aspects are associated with recovery and nonrecovery in ACLF., Approach and Results: Here, we mapped the single-cell transcriptome of circulating immune cells from patients with ACLF and acute decompensated (AD) cirrhosis and healthy individuals. We further interrogate how these findings, as well as immunometabolic and functional profiles, associate with ACLF-recovery (ACLF-R) or nonrecovery (ACLF-NR). Our analysis unveiled 2 distinct states of classical monocytes (cMons). Hereto, ACLF-R cMons were characterized by transcripts associated with immune and stress tolerance, including anti-inflammatory genes such as RETN and LGALS1 . Additional metabolomic and functional validation experiments implicated an elevated oxidative phosphorylation metabolic program as well as an impaired ACLF-R cMon functionality. Interestingly, we observed a common stress-induced tolerant state, oxidative phosphorylation program, and blunted activation among lymphoid populations in patients with ACLF-R. Conversely, ACLF-NR cMon featured elevated expression of inflammatory and stress response genes such as VIM , LGALS2 , and TREM1 , along with blunted metabolic activity and increased functionality., Conclusions: This study identifies distinct immunometabolic cellular states that contribute to disease outcomes in patients with ACLF. Our findings provide valuable insights into the pathogenesis of ACLF, shedding light on factors driving either recovery or nonrecovery phenotypes, which may be harnessed as potential therapeutic targets in the future., (Copyright © 2024 The Author(s). Published by Wolters Kluwer Health, Inc.)

Congenital disorders of glycosylation (CDG) are a large family of rare disorders affecting the different glycosylation pathways. Defective glycosylation can affect any organ, with varying symptoms among the different CDG. Even between individuals with the same CDG there is quite variable severity. Associating specific symptoms to deficiencies of certain glycoproteins or glycolipids is thus a challenging task. In this review, we focus on the glycosphingolipid (GSL) synthesis pathway, which is still rather unexplored in the context of CDG, and outline the functions of the main GSLs, including gangliosides, and their role in the central nervous system. We provide an overview of GSL studies that have been performed in CDG and show that abnormal GSL levels are not only observed in CDG directly affecting GSL synthesis, but also in better known CDG, such as PMM2-CDG. We highlight the importance of studying GSLs in CDG in order to better understand the pathophysiology of these disorders., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is a rare inborn error of metabolism caused by deficiency of the PMM2 enzyme, which leads to impaired protein glycosylation. While the disorder presents with primarily neurological symptoms, there is limited knowledge about the specific brain-related changes caused by PMM2 deficiency. Here, we demonstrate aberrant neural activity in 2D neuronal networks from PMM2-CDG individuals. Utilizing multi-omics datasets from 3D human cortical organoids (hCOs) derived from PMM2-CDG individuals, we identify widespread decreases in protein glycosylation, highlighting impaired glycosylation as a key pathological feature of PMM2-CDG, as well as impaired mitochondrial structure and abnormal glucose metabolism in PMM2-deficient hCOs, indicating disturbances in energy metabolism. Correlation between PMM2 enzymatic activity in hCOs and symptom severity suggests that the level of PMM2 enzyme function directly influences neurological manifestations. These findings enhance our understanding of specific brain-related perturbations associated with PMM2-CDG, offering insights into the underlying mechanisms and potential directions for therapeutic interventions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

T cell exhaustion is a hallmark of cancer and persistent infections, marked by inhibitory receptor upregulation, diminished cytokine secretion, and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex), but the metabolic principles governing Tpex maintenance and the regulatory circuits that control their exhaustion remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics, and metabolomic analyses, we show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the functional exhaustion of T cells. At the molecular level, we find that mitochondrial dysfunction causes redox stress, which inhibits the proteasomal degradation of hypoxia-inducible factor 1α (HIF-1α) and promotes the transcriptional and metabolic reprogramming of Tpex cells into terminally exhausted T cells. Our findings also bear clinical significance, as metabolic engineering of chimeric antigen receptor (CAR) T cells is a promising strategy to enhance the stemness and functionality of Tpex cells for cancer immunotherapy., (© 2023. The Author(s).)

Introduction: Naïve T cells remain in an actively maintained state of quiescence until activation by antigenic signals, upon which they start to proliferate and generate effector cells to initiate a functional immune response. Metabolic reprogramming is essential to meet the biosynthetic demands of the differentiation process, and failure to do so can promote the development of hypofunctional exhausted T cells., Methods: Here we used 13C metabolomics and transcriptomics to study the metabolism of CD8+ T cells in their complete course of differentiation from naïve over stem-like memory to effector cells and in exhaustion-inducing conditions., Results: The quiescence of naïve T cells was evident in a profound suppression of glucose oxidation and a decreased expression of ENO1, downstream of which no glycolytic flux was detectable. Moreover, TCA cycle activity was low in naïve T cells and associated with a downregulation of SDH subunits. Upon stimulation and exit from quiescence, the initiation of cell growth and proliferation was accompanied by differential expression of metabolic enzymes and metabolic reprogramming towards aerobic glycolysis with high rates of nutrient uptake, respiration and lactate production. High flux in anabolic pathways imposed a strain on NADH homeostasis, which coincided with engagement of the proline cycle for mitochondrial redox shuttling. With acquisition of effector functions, cells increasingly relied on glycolysis as opposed to oxidative phosphorylation, which was, however, not linked to changes in mitochondrial abundance. In exhaustion, decreased effector function concurred with a reduction in mitochondrial metabolism, glycolysis and amino acid import, and an upregulation of quiescence-associated genes, TXNIP and KLF2, and the T cell suppressive metabolites succinate and itaconate., Discussion: Overall, these results identify multiple metabolic features that regulate quiescence, proliferation and effector function, but also exhaustion of CD8+ T cells during differentiation. Thus, targeting these metabolic checkpoints may be a promising therapeutic strategy for both prevention of exhaustion and promotion of stemness of anti-tumor T cells., Competing Interests: Authors AnS and PH were employed by Tyrol Clinics GmbH. The remaining 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 © 2023 Kirchmair, Nemati, Lamberti, Trefny, Krogsdam, Siller, Hörtnagl, Schumacher, Sopper, Sandbichler, Zippelius, Ghesquière and Trajanoski.)

Intracellular Ca 2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca 2+ is intracellular BAPTA (BAPTA i ), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTA i enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca 2+ signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTA i enhances cell death in B-cell cancers. In this study, we discovered that BAPTA i alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTA i provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTA i diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTA i . Notably, these effects were also induced by a BAPTA i analog with low affinity for Ca 2+ . Consequently, our findings uncover PFKFB3 inhibition as an Ca 2+ -independent mechanism through which BAPTA i impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTA i are not necessarily related to Ca 2+ signaling. Our data support the need for a reassessment of the role of Ca 2+ in cellular processes when findings were based on the use of BAPTA i ., (© 2023. The Author(s).)

Protective immunity against pathogens or cancer is mediated by the activation and clonal expansion of antigen-specific naive T cells into effector T cells. To sustain their rapid proliferation and effector functions, naive T cells switch their quiescent metabolism to an anabolic metabolism through increased levels of aerobic glycolysis, but also through mitochondrial metabolism and oxidative phosphorylation, generating energy and signalling molecules 1-3 . However, how that metabolic rewiring drives and defines the differentiation of T cells remains unclear. Here we show that proliferating effector CD8 + T cells reductively carboxylate glutamine through the mitochondrial enzyme isocitrate dehydrogenase 2 (IDH2). Notably, deletion of the gene encoding IDH2 does not impair the proliferation of T cells nor their effector function, but promotes the differentiation of memory CD8 + T cells. Accordingly, inhibiting IDH2 during ex vivo manufacturing of chimeric antigen receptor (CAR) T cells induces features of memory T cells and enhances antitumour activity in melanoma, leukaemia and multiple myeloma. Mechanistically, inhibition of IDH2 activates compensating metabolic pathways that cause a disequilibrium in metabolites regulating histone-modifying enzymes, and this maintains chromatin accessibility at genes that are required for the differentiation of memory T cells. These findings show that reductive carboxylation in CD8 + T cells is dispensable for their effector response and proliferation, but that it mainly produces a pattern of metabolites that epigenetically locks CD8 + T cells into a terminal effector differentiation program. Blocking this metabolic route allows the increased formation of memory T cells, which could be exploited to optimize the therapeutic efficacy of CAR T cells., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Malaria remains a widespread health problem with a huge burden. Severe or complicated malaria is highly lethal and encompasses a variety of pathological processes, including immune activation, inflammation, and dysmetabolism. Previously, we showed that adrenal hormones, in particular glucocorticoids (GCs), play critical roles to maintain disease tolerance during Plasmodium infection in mice. Here, GC responses were studied in Cameroon in children with uncomplicated malaria (UM), severe malaria (SM) and asymptomatic controls (AC)., Methods: To determine the sensitivity of leukocytes to GC signaling on a transcriptional level, we measured the ex vivo induction of glucocorticoid induced leucine zipper (GILZ) and FK506-binding protein 5 (FKBP5) by GCs in human and murine leukocytes. Targeted tracer metabolomics on peripheral blood mononuclear cells (PBMCs) was performed to detect metabolic changes induced by GCs., Results: Total cortisol levels increased in patients with clinical malaria compared to AC and were higher in the SM versus UM group, while cortisol binding globulin levels were unchanged and adrenocorticotropic hormone (ACTH) levels were heterogeneous. Induction of both GILZ and FKBP5 by GCs was significantly reduced in patients with clinical malaria compared to AC and in malaria-infected mice compared to uninfected controls. Increased activity in the pentose phosphate pathway was found in the patients, but this was not affected by ex vivo stimulation with physiological levels of hydrocortisone. Interestingly, hydrocortisone induced increased levels of cAMP in AC, but not in clinical malaria patients., Discussion: Altogether, this study shows that patients with SM have increased cortisol levels, but also a decreased sensitivity to GCs, which may clearly contribute to the severity of disease., 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 Vandermosten, Prenen, Fogang, Dagneau de Richecour, Knoops, Donkeu, Nguefack, Taguebue, Ndombo, Ghesquière, Ayong and Van den Steen.)

Abnormal polyol metabolism is predominantly associated with diabetes, where excess glucose is converted to sorbitol by aldose reductase (AR). Recently, abnormal polyol metabolism has been implicated in phosphomannomutase 2 congenital disorder of glycosylation (PMM2-CDG) and an AR inhibitor, epalrestat, proposed as a potential therapy. Considering that the PMM2 enzyme is not directly involved in polyol metabolism, the increased polyol production and epalrestat's therapeutic mechanism in PMM2-CDG remained elusive. PMM2-CDG, caused by PMM2 deficiency, presents with depleted GDP-mannose and abnormal glycosylation. Here, we show that, apart from glycosylation abnormalities, PMM2 deficiency affects intracellular glucose flux, resulting in polyol increase. Targeting AR with epalrestat decreases polyols and increases GDP-mannose both in patient-derived fibroblasts and in pmm2 mutant zebrafish. Using tracer studies, we demonstrate that AR inhibition diverts glucose flux away from polyol production toward the synthesis of sugar nucleotides, and ultimately glycosylation. Finally, PMM2-CDG individuals treated with epalrestat show a clinical and biochemical improvement., Competing Interests: Declaration of interests Mayo Clinic and E.M. have a financial interest related to this research. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and is being conducted in compliance with Mayo Clinic Conflict of Interest policies. E.M. has the following patents planned, issued, or pending: application title, “Methods and Materials for Treating Glycosylation Disorders”; application # 16/973,210; filing date, 12/08/2020; Mayo Case # 2018-132., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

This scoping review summarizes what is known about kidney metabolism during hypothermic perfusion preservation. Papers studying kidney metabolism during hypothermic (<12 °C) perfusion were identified (PubMed, Embase, Web of Science, Cochrane). Out of 14,335 initially identified records, 52 were included [dog (26/52), rabbit (2/52), pig (20/52), human (7/52)]. These were published between 1970-2023, partially explaining study heterogeneity. There is a considerable risk of bias in the reported studies. Studies used different perfusates, oxygenation levels, kidney injury levels, and devices and reported on perfusate and tissue metabolites. In 11 papers, (non)radioactively labeled metabolites (tracers) were used to study metabolic pathways. Together these studies show that kidneys are metabolically active during hypothermic perfusion, regardless of the perfusion setting. Although tracers give us more insight into active metabolic pathways, kidney metabolism during hypothermic perfusion is incompletely understood. Metabolism is influenced by perfusate composition, oxygenation levels, and likely also by pre-existing ischemic injury. In the modern era, with increasing donations after circulatory death and the emergence of hypothermic oxygenated perfusion, the focus should be on understanding metabolic perturbations caused by pre-existing injury levels and the effect of perfusate oxygen levels. The use of tracers is indispensable to understanding the kidney's metabolism during perfusion, given the complexity of interactions between different metabolites.

Neuronal development in the human cerebral cortex is considerably prolonged compared with that of other mammals. We explored whether mitochondria influence the species-specific timing of cortical neuron maturation. By comparing human and mouse cortical neuronal maturation at high temporal and cell resolution, we found a slower mitochondria development in human cortical neurons compared with that in the mouse, together with lower mitochondria metabolic activity, particularly that of oxidative phosphorylation. Stimulation of mitochondria metabolism in human neurons resulted in accelerated development in vitro and in vivo, leading to maturation of cells weeks ahead of time, whereas its inhibition in mouse neurons led to decreased rates of maturation. Mitochondria are thus important regulators of the pace of neuronal development underlying human-specific brain neoteny.

Metabolic reprogramming can contribute to colorectal cancer progression and therapy resistance. Identification of key regulators of colorectal cancer metabolism could provide new approaches to improve treatment and reduce recurrence. Here, we demonstrate a critical role for the COP9 signalosome subunit CSN6 in rewiring nucleotide metabolism in colorectal cancer. Transcriptomic analysis of colorectal cancer patient samples revealed a correlation between CSN6 expression and purine and pyrimidine metabolism. A colitis-associated colorectal cancer model established that Csn6 intestinal conditional deletion decreased tumor development and altered nucleotide metabolism. CSN6 knockdown increased the chemosensitivity of colorectal cancer cells in vitro and in vivo, which could be partially reversed with nucleoside supplementation. Isotope metabolite tracing showed that CSN6 loss reduced de novo nucleotide synthesis. Mechanistically, CSN6 upregulated purine and pyrimidine biosynthesis by increasing expression of PHGDH, a key enzyme in the serine synthesis pathway. CSN6 inhibited β-Trcp-mediated DDX5 polyubiquitination and degradation, which in turn promoted DDX5-mediated PHGDH mRNA stabilization, leading to metabolic reprogramming and colorectal cancer progression. Butyrate treatment decreased CSN6 expression and improved chemotherapy efficacy. These findings unravel the oncogenic role of CSN6 in regulating nucleotide metabolism and chemosensitivity in colorectal cancer., Significance: CSN6 deficiency inhibits colorectal cancer development and chemoresistance by downregulating PHGDH to block nucleotide biosynthesis, providing potential therapeutic targets to improve colorectal cancer treatment., (©2022 American Association for Cancer Research.)

Background: Understanding kidney metabolism during perfusion is vital to further develop the technology as a preservation, viability assessment, and resuscitation platform. We reviewed the evidence on the use of labeled metabolites (tracers) to understand "on-pump" kidney behavior., Methods: PubMed, Embase, Web of Science, and Cochrane databases were systematically searched for studies evaluating metabolism of (non)radioactively labeled endogenous compounds during kidney perfusion., Results: Of 5899 articles, 30 were included. All were animal studies [rat (70%), dog (13%), pig (10%), rabbit (7%)] perfusing but not transplanting kidneys. Perfusion took place at hypothermic (4-12°C) (20%), normothermic (35-40°C) (77%), or undefined temperatures (3%). Hypothermic perfusion used albumin or a clinical kidney preservation solution, mostly in the presence of oxygen. Normothermic perfusion was mostly performed with oxygenated crystalloids often containing glucose and amino acids with unclear partial oxygen tensions. Active metabolism of carbohydrate, amino acid, lipids, and large molecules was shown in hypothermic and normothermic perfusion. Production of macromolecules, such as prostaglandin, thromboxane, and vitamin D, takes place during normothermic perfusion. No experiments compared differences in metabolic activity between hypothermic and normothermic perfusion. One conference abstract showed increased anaerobic metabolism in kidneys donated after circulatory death by adding labeled glucose to hypothermically perfused human kidneys., Conclusions: Tracer studies during kidney perfusion contribute to unraveling kidney metabolic behavior in pre-clinical models. Whether findings are truly translational needs further investigation in large animal models of human kidneys. Furthermore, it is essential to better understand how ischemia changes this metabolic behavior., (© 2022 International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals LLC.)

Hepatic encephalopathy (HE) is a common complication of chronic liver disease, characterized by an altered mental state and hyperammonemia. Insight into the brain pathophysiology of HE is limited due to a paucity of well-characterized HE models beyond the rat bile duct ligation (BDL) model. Here, we assess the presence of HE characteristics in the mouse BDL model. We show that BDL in C57Bl/6j mice induces motor dysfunction, progressive liver fibrosis, liver function failure and hyperammonemia, all hallmarks of HE. Swiss mice however fail to replicate the same phenotype, underscoring the importance of careful strain selection. Next, in-depth characterisation of metabolic disturbances in the cerebrospinal fluid of BDL mice shows glutamine accumulation and transient decreases in taurine and choline, indicative of brain ammonia overload. Moreover, mouse BDL induces glial cell dysfunction, namely microglial morphological changes with neuroinflammation and astrocyte reactivity with blood-brain barrier (BBB) disruption. Finally, we identify putative novel mechanisms involved in central HE pathophysiology, like bile acid accumulation and tryptophan-kynurenine pathway alterations. Our study provides the first comprehensive evaluation of a mouse model of HE in chronic liver disease. Additionally, this study further underscores the importance of neuroinflammation in the central effects of chronic liver disease., (© 2022. The Author(s).)

Introduction: Primary mitochondrial diseases (PMD) are a large, heterogeneous group of genetic disorders affecting mitochondrial function, mostly by disrupting the oxidative phosphorylation (OXPHOS) system. Understanding the cellular metabolic re-wiring occurring in PMD is crucial for the development of novel diagnostic tools and treatments, as PMD are often complex to diagnose and most of them currently have no effective therapy., Objectives: To characterize the cellular metabolic consequences of OXPHOS dysfunction and based on the metabolic signature, to design new diagnostic and therapeutic strategies., Methods: In vitro assays were performed in skin-derived fibroblasts obtained from patients with diverse PMD and validated in pharmacological models of OXPHOS dysfunction. Proliferation was assessed using the Incucyte technology. Steady-state glucose and glutamine tracing studies were performed with LC-MS quantification of cellular metabolites. The therapeutic potential of nutritional supplements was evaluated by assessing their effect on proliferation and on the metabolomics profile. Successful therapies were then tested in a in vivo lethal rotenone model in zebrafish., Results: OXPHOS dysfunction has a unique metabolic signature linked to an NAD+/NADH imbalance including depletion of TCA intermediates and aspartate, and increased levels of glycerol-3-phosphate. Supplementation with pyruvate and uridine fully rescues this altered metabolic profile and the subsequent proliferation deficit. Additionally, in zebrafish, the same nutritional treatment increases the survival after rotenone exposure., Conclusions: Our findings reinforce the importance of the NAD+/NADH imbalance following OXPHOS dysfunction in PMD and open the door to new diagnostic and therapeutic tools for PMD., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

A functional electron transport chain (ETC) is crucial for supporting bioenergetics and biosynthesis. Accordingly, ETC inhibition decreases proliferation in cancer cells but does not seem to impair stem cell proliferation. However, it remains unclear how stem cells metabolically adapt. In this study, we show that pharmacological inhibition of complex III of the ETC in skeletal stem and progenitor cells induces glycolysis side pathways and reroutes the tricarboxylic acid (TCA) cycle to regenerate NAD + and preserve cell proliferation. These metabolic changes also culminate in increased succinate and 2-hydroxyglutarate levels that inhibit Ten-eleven translocation (TET) DNA demethylase activity, thereby preserving self-renewal and multilineage potential. Mechanistically, mitochondrial malate dehydrogenase and reverse succinate dehydrogenase activity proved to be essential for the metabolic rewiring in response to ETC inhibition. Together, these data show that the metabolic plasticity of skeletal stem and progenitor cells allows them to bypass ETC blockade and preserve their self-renewal., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Tracer metabolomics is a powerful technology for the biomedical community to study and understand disease-inflicted metabolic mechanisms. However, the interpretation of tracer metabolomics results is highly technical, as the metabolites' abundances, tracer incorporation and positions on the metabolic map all must be jointly interpreted. The field is currently lacking a structured approach to help less experienced researchers start the interpretation of tracer metabolomics datasets. We propose an approach using an intuitive visualization concept aided by a novel open-source tool, and provide guidelines on how researchers can apply the approach and the visualization tool to their own datasets. Using a showcase experiment, we demonstrate that the visualization approach leads to an intuitive interpretation that can ease researchers into understanding their tracer metabolomics data.

Core facilities have a different mission than academic research labs. Accordingly, they require different career paths and structures., (© 2022 The Authors.)

Autophagy has vasculoprotective roles, but whether and how it regulates lymphatic endothelial cells (LEC) homeostasis and lymphangiogenesis is unknown. Here, we show that genetic deficiency of autophagy in LEC impairs responses to VEGF-C and injury-driven corneal lymphangiogenesis. Autophagy loss in LEC compromises the expression of main effectors of LEC identity, like VEGFR3, affects mitochondrial dynamics and causes an accumulation of lipid droplets (LDs) in vitro and in vivo. When lipophagy is impaired, mitochondrial ATP production, fatty acid oxidation, acetyl-CoA/CoA ratio and expression of lymphangiogenic PROX1 target genes are dwindled. Enforcing mitochondria fusion by silencing dynamin-related-protein 1 (DRP1) in autophagy-deficient LEC fails to restore LDs turnover and lymphatic gene expression, whereas supplementing the fatty acid precursor acetate rescues VEGFR3 levels and signaling, and lymphangiogenesis in LEC-Atg5 -/- mice. Our findings reveal that lipophagy in LEC by supporting FAO, preserves a mitochondrial-PROX1 gene expression circuit that safeguards LEC responsiveness to lymphangiogenic mediators and lymphangiogenesis., (© 2022. The Author(s).)

Metabolic reprogramming is a hallmark of activated T cells. The switch from oxidative phosphorylation to aerobic glycolysis provides energy and intermediary metabolites for the biosynthesis of macromolecules to support clonal expansion and effector function. Here, we show that glycolytic reprogramming additionally controls inflammatory gene expression via epigenetic remodeling. We found that the glucose transporter GLUT3 is essential for the effector functions of Th17 cells in models of autoimmune colitis and encephalomyelitis. At the molecular level, we show that GLUT3-dependent glucose uptake controls a metabolic-transcriptional circuit that regulates the pathogenicity of Th17 cells. Metabolomic, epigenetic, and transcriptomic analyses linked GLUT3 to mitochondrial glucose oxidation and ACLY-dependent acetyl-CoA generation as a rate-limiting step in the epigenetic regulation of inflammatory gene expression. Our findings are also important from a translational perspective because inhibiting GLUT3-dependent acetyl-CoA generation is a promising metabolic checkpoint to mitigate Th17-cell-mediated inflammatory diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Molecular drivers of metastasis in patients with high-risk localized prostate cancer are poorly understood. Therefore, we aim to study molecular drivers of metastatic progression in patients with high-risk prostate cancer. A retrospective matched case-control study of two clinico-pathologically identical groups of patients with high-risk prostate cancer was undertaken. One group developed metastatic recurrence (n = 19) while the other did not (n = 25). The primary index tumor was identified by a uro-pathologist, followed by DNA and RNA extraction for somatic copy-number aberration (SCNA) analysis and whole-transcriptome gene expression analysis. In vitro and in vivo studies included cell line manipulation and xenograft models., The integrative CNA and gene expression analyses identified an increase in Antizyme Inhibitor 1 (AZIN1) gene expression within a focal amplification of 8q22.3, which was associated with metastatic recurrence of patients with high-risk prostate cancer in four independent cohorts. The effects of AZIN1 knockdown were evaluated, due to its therapeutic potential. AZIN1 knockdown effected proliferation and metastatic potential of prostate cancer cells and xenograft models. RNA sequencing after AZIN1 knockdown in prostate cancer cells revealed upregulation of genes coding for collagen subunits. The observed effect on cell migration after AZIN1 knockdown was mimicked when exposing prostate cancer cells to bio-active molecules deriving from COL4A1 and COL4A2. Our integrated CNA and gene expression analysis of primary high-risk prostate cancer identified the AZIN1 gene as a novel driver of metastatic progression, by altering collagen subunit expression. Future research should further investigate its therapeutic potential in preventing metastatic recurrence., Implications: AZIN1 was identified as driver of metastatic progression in high-risk prostate cancer through matrikine regulation., (©2022 American Association for Cancer Research.)

Purpose: TRAPPC9 deficiency is an autosomal recessive disorder mainly associated with intellectual disability (ID), microcephaly, and obesity. Previously, TRAPPC9 deficiency has not been associated with biochemical abnormalities., Methods: Exome sequencing was performed in 3 individuals with ID and dysmorphic features. N-Glycosylation analyses were performed in the patients' blood samples to test for possible congenital disorder of glycosylation (CDG). TRAPPC9 gene, TRAPPC9 protein expression, and N-glycosylation markers were assessed in patient fibroblasts. Complementation with wild-type TRAPPC9 and immunofluorescence studies to assess TRAPPC9 expression and localization were performed. The metabolic consequences of TRAPPC9 deficiency were evaluated using tracer metabolomics., Results: All 3 patients carried biallelic missense variants in TRAPPC9 and presented with an N-glycosylation defect in blood, consistent with CDG type I. Extensive investigations in patient fibroblasts corroborated TRAPPC9 deficiency and an N-glycosylation defect. Tracer metabolomics revealed global metabolic changes with several affected glycosylation-related metabolites., Conclusion: We identified 3 TRAPPC9 deficient patients presenting with ID, dysmorphic features, and abnormal glycosylation. On the basis of our findings, we propose that TRAPPC9 deficiency could lead to a CDG (TRAPPC9-CDG). The finding of abnormal glycosylation in these patients is highly relevant for diagnosis, further elucidation of the pathophysiology, and management of the disease., Competing Interests: Conflicts of Interest Authors declare no conflicts of interests., (Copyright © 2021 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Background: Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both MM cell survival and drug resistance. Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are two mitochondrial enzymes that facilitate the last step in the glutamine-to-proline conversion. Overexpression of PYCR1 is involved in progression of several cancers, however, its' role in hematological cancers is unknown. In this study, we investigated whether PYCR affects MM viability, proliferation and response to bortezomib., Methods: Correlation of PYCR1/2 with overall survival was investigated in the MMRF CoMMpass trial (653 patients). OPM-2 and RPMI-8226 MM cell lines were used to perform in vitro experiments. RPMI-8226 cells were supplemented with 13 C-glutamine for 48 h in both normoxia and hypoxia (< 1% O 2 , by chamber) to perform a tracer study. PYCR1 was inhibited by siRNA or the small molecule inhibitor pargyline. Apoptosis was measured using Annexin V and 7-AAD staining, viability by CellTiterGlo assay and proliferation by BrdU incorporation. Differential protein expression was evaluated using Western Blot. The SUnSET method was used to measure protein synthesis. All in vitro experiments were performed in hypoxic conditions., Results: We found that PYCR1 and PYCR2 mRNA expression correlated with an inferior overall survival. MM cells from relapsed/refractory patients express significantly higher levels of PYCR1 mRNA. In line with the strong expression of PYCR1, we performed a tracer study in RPMI-8226 cells, which revealed an increased conversion of 13 C-glutamine to proline in hypoxia. PYCR1 inhibition reduced MM viability and proliferation and increased apoptosis. Mechanistically, we found that PYCR1 silencing reduced protein levels of p-PRAS40, p-mTOR, p-p70, p-S6, p-4EBP1 and p-eIF4E levels, suggesting a decrease in protein synthesis, which we also confirmed in vitro. Pargyline and siPYCR1 increased bortezomib-mediated apoptosis. Finally, combination therapy of pargyline with bortezomib reduced viability in CD138 +  MM cells and reduced tumor burden in the murine 5TGM1 model compared to single agents., Conclusions: This study identifies PYCR1 as a novel target in bortezomib-based combination therapies for MM., (© 2022. The Author(s).)

Here, we investigate the impact of hypoxia on the hepatic response of glucocorticoid receptor (GR) to dexamethasone (DEX) in mice via RNA-sequencing. Hypoxia causes three types of reprogramming of GR: (i) much weaker induction of classical GR-responsive genes by DEX in hypoxia, (ii) a number of genes is induced by DEX specifically in hypoxia, and (iii) hypoxia induces a group of genes via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Transcriptional profiles are reflected by changed GR DNA-binding as measured by ChIP sequencing. The HPA axis is induced by hypothalamic HIF1α and HIF2α activation and leads to GR-dependent lipolysis and ketogenesis. Acute inflammation, induced by lipopolysaccharide, is prevented by DEX in normoxia but not during hypoxia, and this is attributed to HPA axis activation by hypoxia. We unfold new physiological pathways that have consequences for patients suffering from GC resistance., (© 2021 The Authors.)

Objective: Epalrestat, an aldose reductase inhibitor increases phosphomannomutase (PMM) enzyme activity in a PMM2-congenital disorders of glycosylation (CDG) worm model. Epalrestat also decreases sorbitol level in diabetic neuropathy. We evaluated the genetic, biochemical, and clinical characteristics, including the Nijmegen Progression CDG Rating Scale (NPCRS), urine polyol levels and fibroblast glycoproteomics in patients with PMM2-CDG., Methods: We performed PMM enzyme measurements, multiplexed proteomics, and glycoproteomics in PMM2-deficient fibroblasts before and after epalrestat treatment. Safety and efficacy of 0.8 mg/kg/day oral epalrestat were studied in a child with PMM2-CDG for 12 months., Results: PMM enzyme activity increased post-epalrestat treatment. Compared with controls, 24% of glycopeptides had reduced abundance in PMM2-deficient fibroblasts, 46% of which improved upon treatment. Total protein N-glycosylation improved upon epalrestat treatment bringing overall glycosylation toward the control fibroblasts' glycosylation profile. Sorbitol levels were increased in the urine of 74% of patients with PMM2-CDG and correlated with the presence of peripheral neuropathy, and CDG severity rating scale. In the child with PMM2-CDG on epalrestat treatment, ataxia scores improved together with significant growth improvement. Urinary sorbitol levels nearly normalized in 3 months and blood transferrin glycosylation normalized in 6 months., Interpretation: Epalrestat improved PMM enzyme activity, N-glycosylation, and glycosylation biomarkers in vitro. Leveraging cellular glycoproteome assessment, we provided a systems-level view of treatment efficacy and discovered potential novel biosignatures of therapy response. Epalrestat was well-tolerated and led to significant clinical improvements in the first pediatric patient with PMM2-CDG treated with epalrestat. We also propose urinary sorbitol as a novel biomarker for disease severity and treatment response in future clinical trials in PMM2-CDG. ANN NEUROL 20219999:n/a-n/a., (© 2021 American Neurological Association.)