Western diet increases brain metabolism and adaptive immune responses in a mouse model of amyloidosis.

Diet-induced increase in body weight is a growing health concern worldwide. Often accompanied by a low-grade metabolic inflammation that changes systemic functions, diet-induced alterations may contribute to neurodegenerative disorder progression as well. This study aims to non-invasively investigate diet-induced metabolic and inflammatory effects in the brain of an APPPS1 mouse model of Alzheimer's disease. [18F]FDG, [18F]FTHA, and [18F]GE-180 were used for in vivo PET imaging in wild-type and APPPS1 mice. Ex vivo flow cytometry and histology in brains complemented the in vivo findings. 1H- magnetic resonance spectroscopy in the liver, plasma metabolomics and flow cytometry of the white adipose tissue were used to confirm metaflammatory condition in the periphery. We found disrupted glucose and fatty acid metabolism after Western diet consumption, with only small regional changes in glial-dependent neuroinflammation in the brains of APPPS1 mice. Further ex vivo investigations revealed cytotoxic T cell involvement in the brains of Western diet-fed mice and a disrupted plasma metabolome. 1H-magentic resonance spectroscopy and immunological results revealed diet-dependent inflammatory-like misbalance in livers and fatty tissue. Our multimodal imaging study highlights the role of the brain-liver-fat axis and the adaptive immune system in the disruption of brain homeostasis in amyloid models of Alzheimer's disease.

A 5:2 intermittent fasting regimen ameliorates NASH and fibrosis and blunts HCC development via hepatic PPARα and PCK1.

The role and molecular mechanisms of intermittent fasting (IF) in non-alcoholic steatohepatitis (NASH) and its transition to hepatocellular carcinoma (HCC) are unknown. Here, we identified that an IF 5:2 regimen prevents NASH development as well as ameliorates established NASH and fibrosis without affecting total calorie intake. Furthermore, the IF 5:2 regimen blunted NASH-HCC transition when applied therapeutically. The timing, length, and number of fasting cycles as well as the type of NASH diet were critical parameters determining the benefits of fasting. Combined proteome, transcriptome, and metabolome analyses identified that peroxisome-proliferator-activated receptor alpha (PPARα) and glucocorticoid-signaling-induced PCK1 act co-operatively as hepatic executors of the fasting response. In line with this, PPARα targets and PCK1 were reduced in human NASH. Notably, only fasting initiated during the active phase of mice robustly induced glucocorticoid signaling and free-fatty-acid-induced PPARα signaling. However, hepatocyte-specific glucocorticoid receptor deletion only partially abrogated the hepatic fasting response. In contrast, the combined knockdown of Ppara and Pck1 in vivo abolished the beneficial outcomes of fasting against inflammation and fibrosis. Moreover, overexpression of Pck1 alone or together with Ppara in vivo lowered hepatic triglycerides and steatosis. Our data support the notion that the IF 5:2 regimen is a promising intervention against NASH and subsequent liver cancer.

Retinal metabolism displays evidence for uncoupling of glycolysis and oxidative phosphorylation via Cori-, Cahill-, and mini-Krebs-cycle.

The retina consumes massive amounts of energy, yet its metabolism and substrate exploitation remain poorly understood. Here, we used a murine explant model to manipulate retinal energy metabolism under entirely controlled conditions and utilised 1H-NMR spectroscopy-based metabolomics, in situ enzyme detection, and cell viability readouts to uncover the pathways of retinal energy production. Our experimental manipulations resulted in varying degrees of photoreceptor degeneration, while the inner retina and retinal pigment epithelium were essentially unaffected. This selective vulnerability of photoreceptors suggested very specific adaptations in their energy metabolism. Rod photoreceptors were found to rely strongly on oxidative phosphorylation, but only mildly on glycolysis. Conversely, cone photoreceptors were dependent on glycolysis but insensitive to electron transport chain decoupling. Importantly, photoreceptors appeared to uncouple glycolytic and Krebs-cycle metabolism via three different pathways: (1) the mini-Krebs-cycle, fuelled by glutamine and branched chain amino acids, generating N-acetylaspartate; (2) the alanine-generating Cahill-cycle; (3) the lactate-releasing Cori-cycle. Moreover, the metabolomics data indicated a shuttling of taurine and hypotaurine between the retinal pigment epithelium and photoreceptors, likely resulting in an additional net transfer of reducing power to photoreceptors. These findings expand our understanding of retinal physiology and pathology and shed new light on neuronal energy homeostasis and the pathogenesis of neurodegenerative diseases.

Metabolic fingerprinting by nuclear magnetic resonance of hepatocellular carcinoma cells during p53 reactivation-induced senescence.

Cellular senescence is characterized by stable cell cycle arrest. Senescent cells exhibit a senescence-associated secretory phenotype that can promote tumor progression. The aim of our study was to identify specific nuclear magnetic resonance (NMR) spectroscopy-based markers of cancer cell senescence. For metabolic studies, we employed murine liver carcinoma Harvey Rat Sarcoma Virus (H-Ras) cells, in which reactivation of p53 expression induces senescence. Senescent and nonsenescent cell extracts were subjected to high-resolution proton (1H)-NMR spectroscopy-based metabolomics, and dynamic metabolic changes during senescence were analyzed using a magnetic resonance spectroscopy (MRS)-compatible cell perfusion system. Additionally, the ability of intact senescent cells to degrade the extracellular matrix (ECM) was quantified in the cell perfusion system. Analysis of senescent H-Ras cell extracts revealed elevated sn-glycero-3-phosphocholine, myoinositol, taurine, and creatine levels, with decreases in glycine, o-phosphocholine, threonine, and valine. These metabolic findings were accompanied by a greater degradation index of the ECM in senescent H-Ras cells than in control H-Ras cells. MRS studies with the cell perfusion system revealed elevated creatine levels in senescent cells on Day 4, confirming the 1H-NMR results. These senescence-associated changes in metabolism and ECM degradation strongly impact growth and redox metabolism and reveal potential MRS signals for detecting senescent cancer cells in vivo.

Twelve-hour normothermic liver perfusion in a rat model: characterization of the changes in the ex-situ bio-molecular phenotype and metabolism.

The partial understanding of the biological events that occur during normothermic machine perfusion (NMP) and particularly during prolonged perfusion might hinder its deployment in clinical transplantation. The aim of our study was to implement a rat model of prolonged NMP to characterize the bio-molecular phenotype and metabolism of the perfused organs. Livers (n = 5/group) were procured and underwent 4 h (NMP4h) or 12 h (NMP12h) NMP, respectively, using a perfusion fluid supplemented with an acellular oxygen carrier. Organs that were not exposed to any procedure served as controls (Native). All perfused organs met clinically derived viability criteria at the end of NMP. Factors related to stress-response and survival were increased after prolonged perfusion. No signs of oxidative damage were detected in both NMP groups. Evaluation of metabolite profiles showed preserved mitochondrial function, activation of Cori cycle, induction of lipolysis, acetogenesis and ketogenesis in livers exposed to 12 h-NMP. Increased concentrations of metabolites involved in glycogen synthesis, glucuronidation, bile acid conjugation, and antioxidant response were likewise observed. In conclusion, our NMP12h model was able to sustain liver viability and function, thereby deeply changing cell homeostasis to maintain a newly developed equilibrium. Our findings provide valuable information for the implementation of optimized protocols for prolonged NMP.

Quantitative Metabolomics and Lipoprotein Analysis of PDAC Patients Suggests Serum Marker Categories for Pancreatic Function, Pancreatectomy, Cancer Metabolism, and Systemic Disturbances.

Pancreatic ductal adenocarcinoma (PDAC) is difficult to diagnose in the early stages and lacks reliable biomarkers. The scope of this project was to establish quantitative nuclear magnetic resonance (NMR) spectroscopy to comprehensively study blood serum alterations in PDAC patients. Serum samples from 34 PDAC patients obtained before and after pancreatectomy as well as 83 age- and sex-matched control samples from healthy donors were analyzed with in vitro diagnostics research (IVDr) proton NMR spectroscopy at 600 MHz. Uni- and multivariate statistics were applied to identify significant biofluid alterations. We identified 29 significantly changed metabolites and 98 lipoproteins when comparing serum from healthy controls with those of PDAC patients. The most prominent features were assigned to (i) markers of pancreatic function (e.g., glucose and blood triglycerides), (ii) markers related to surgery (e.g., ketone bodies and blood cholesterols), (iii) PDAC-associated markers (e.g., amino acids and creatine), and (iv) markers for systemic disturbances in PDAC (e.g., gut metabolites DMG, TMAO, DMSO2, and liver lipoproteins). Quantitative serum NMR spectroscopy is suited as a diagnostic tool to investigate PDAC. Remarkably, 2-hydroxybutyrate (2-HB) as a previously suggested marker for insulin resistance was found in extraordinarily high levels only after pancreatectomy, suggesting this metabolite is the strongest marker for pancreatic loss of function.

Image-guided metabolomics and transcriptomics reveal tumour heterogeneity in luminal A and B human breast cancer beyond glucose tracer uptake.

BACKGROUND: Breast cancer is a metabolically heterogeneous disease, and although the concept of heterogeneous cancer metabolism is known, its precise role in human breast cancer is yet to be fully elucidated. METHODS: We investigated in an explorative approach a cohort of 42 primary mamma carcinoma patients with positron emission tomography/magnetic resonance imaging (PET/MR) prior to surgery, followed by histopathology and molecular diagnosis. From a subset of patients, which showed high metabolic heterogeneity based on tracer uptake and pathology classification, tumour centre and periphery specimen tissue samples were further investigated by a targeted breast cancer gene expression panel and quantitative metabolomics by nuclear magnetic resonance (NMR) spectroscopy. All data were analysed in a combinatory approach. RESULTS: [18 F]FDG (2-deoxy-2-[fluorine-18]fluoro-d-glucose) tracer uptake confirmed dominance of glucose metabolism in the breast tumour centre, with lower levels in the periphery. Additionally, we observed differences in lipid and proliferation related genes between luminal A and B subtypes in the centre and periphery. Tumour periphery showed elevated acetate levels and enrichment in lipid metabolic pathways genes especially in luminal B. Furthermore, serine was increased in the periphery and higher expression of thymidylate synthase (TYMS) indicated one-carbon metabolism increased in tumour periphery. The overall metabolic activity based on [18 F]FDG uptake of luminal B subtype was higher than that of luminal A and the difference between the periphery and centre increased with tumour grade. CONCLUSION: Our analysis indicates variations in metabolism among different breast cancer subtypes and sampling locations which details the heterogeneity of the breast tumours. Correlation analysis of [18 F]FDG tracer uptake, transcriptome and tumour metabolites like acetate and serine facilitate the search for new candidates for metabolic tracers and permit distinguishing luminal A and B. This knowledge may help to differentiate subtypes preclinically or to provide patients guide for neoadjuvant therapy and optimised surgical protocols based on individual tumour metabolism.

Integrative Molecular Structure Elucidation and Construction of an Extended Metabolic Pathway Associated with an Ancient Innate Immune Response in COVID-19 Patients.

We present compelling evidence for the existence of an extended innate viperin-dependent pathway, which provides crucial evidence for an adaptive response to viral agents, such as SARS-CoV-2. We show the in vivo biosynthesis of a family of novel endogenous cytosine metabolites with potential antiviral activities. Two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed a characteristic spin-system motif, indicating the presence of an extended panel of urinary metabolites during the acute viral replication phase. Mass spectrometry additionally enabled the characterization and quantification of the most abundant serum metabolites, showing the potential diagnostic value of the compounds for viral infections. In total, we unveiled ten nucleoside (cytosine- and uracil-based) analogue structures, eight of which were previously unknown in humans allowing us to propose a new extended viperin pathway for the innate production of antiviral compounds. The molecular structures of the nucleoside analogues and their correlation with an array of serum cytokines, including IFN-α2, IFN-γ, and IL-10, suggest an association with the viperin enzyme contributing to an ancient endogenous innate immune defense mechanism against viral infection.

cGMP modulates hemin-mediated platelet death.

BACKGROUND AND AIMS: Hemolysis is a known risk factor for thrombosis resulting in critical limb ischemia and microcirculatory disturbance and organ failure. Intravasal hemolysis may lead to life-threatening complications due to uncontrolled thrombo-inflammation. Until now, conventional antithrombotic therapies failed to control development and progression of these thrombotic events. Thus, the pathophysiology of these thrombotic events needs to be investigated to unravel underlying pathways and thereby identify targets for novel treatment strategies. METHODS: Here we used classical experimental set-ups as well as high-end flow cytometry, metabolomics and lipidomic analysis to in-depth analyze the effects of hemin on platelet physiology and morphology. RESULTS: Hemin does strongly and swiftly induce platelet activation and this process is modulated by the sGC-cGMP-cGKI signaling axis. cGMP modulation also reduced the pro-aggregatory potential of plasma derived from patients with hemolysis. Furthermore, hemin-induced platelet death evokes distinct platelet subpopulations. Typical cell death markers, such as ROS, were induced by hemin-stimulation and the platelet lipidome was specifically altered by high hemin concentration. Specifically, arachidonic acid derivates, such as PGE2, TXB2 or 12-HHT, were significantly increased. Balancing the cGMP levels by modulation of the sGC-cGMP-cGKI axis diminished the ferroptotic effect of hemin. CONCLUSION: We found that cGMP modulates hemin-induced platelet activation and thrombus formation in vitro and cGMP effects hemin-mediated platelet death and changes in the platelet lipidome. Thus, it is tempting to speculate that modulating platelet cGMP levels may be a novel strategy to control thrombosis and critical limb ischemia in patients with hemolytic crisis.

Urinary phenotyping of SARS-CoV-2 infection connects clinical diagnostics with metabolomics and uncovers impaired NAD+ pathway and SIRT1 activation.

OBJECTIVES: The stratification of individuals suffering from acute and post-acute SARS-CoV-2 infection remains a critical challenge. Notably, biomarkers able to specifically monitor viral progression, providing details about patient clinical status, are still not available. Herein, quantitative metabolomics is progressively recognized as a useful tool to describe the consequences of virus-host interactions considering also clinical metadata. METHODS: The present study characterized the urinary metabolic profile of 243 infected individuals by quantitative nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography mass spectrometry (LC-MS). Results were compared with a historical cohort of noninfected subjects. Moreover, we assessed the concentration of recently identified antiviral nucleosides and their association with other metabolites and clinical data. RESULTS: Urinary metabolomics can stratify patients into classes of disease severity, with a discrimination ability comparable to that of clinical biomarkers. Kynurenines showed the highest fold change in clinically-deteriorated patients and higher-risk subjects. Unique metabolite clusters were also generated based on age, sex, and body mass index (BMI). Changes in the concentration of antiviral nucleosides were associated with either other metabolites or clinical variables. Increased kynurenines and reduced trigonelline excretion indicated a disrupted nicotinamide adenine nucleotide (NAD+) and sirtuin 1 (SIRT1) pathway. CONCLUSIONS: Our results confirm the potential of urinary metabolomics for noninvasive diagnostic/prognostic screening and show that the antiviral nucleosides could represent novel biomarkers linking viral load, immune response, and metabolism. Moreover, we established for the first time a casual link between kynurenine accumulation and deranged NAD+/SIRT1, offering a novel mechanism through which SARS-CoV-2 manipulates host physiology.

Stratification of hypertension and SARS-CoV-2 infection by quantitative NMR spectroscopy of human blood serum.

BACKGROUND: Diagnostic approaches like the nuclear magnetic resonance spectroscopy (NMR) based quantification of metabolites, lipoproteins, and inflammation markers has helped to identify typical alterations in the blood serum of COVID-19 patients. However, confounders such as sex, and comorbidities, which strongly influence the metabolome, were often not considered. Therefore, the aim of this NMR study was to consider sex, as well as arterial hypertension (AHT), when investigating COVID-19-positive serum samples in a large age-and sex matched cohort. METHODS: NMR serum data from 329 COVID-19 patients were compared with 305 healthy controls. 134 COVID-19 patients were affected by AHT. These were analyzed together with NMR data from 58 hypertensives without COVID-19. In addition to metabolite, lipoprotein, and glycoprotein data from NMR, common laboratory parameters were considered. Sex was considered in detail for all comparisons. RESULTS: Here, we show that several differences emerge from previous NMR COVID-19 studies when AHT is considered. Especially, the previously described triglyceride-rich lipoprotein profile is no longer observed in COVID-19 patients, nor an increase in ketone bodies. Further alterations are a decrease in glutamine, leucine, isoleucine, and lysine, citric acid, HDL-4 particles, and total cholesterol. Additionally, hypertensive COVID-19 patients show higher inflammatory NMR parameters than normotensive patients. CONCLUSIONS: We present a more precise picture of COVID-19 blood serum parameters. Accordingly, considering sex and comorbidities should be included in future metabolomics studies for improved and refined patient stratification. Due to metabolic similarities with other viral infections, these results can be applied to other respiratory diseases in the future.

The functionality of our human body is driven by a large number of small molecules, called metabolites. These metabolites can be associated with health but also disease conditions. In this study, we used a technology called nuclear magnetic resonance spectroscopy (NMR) to determine metabolite and protein concentrations in the blood of acutely-infected COVID-19 patients and compared these results with disease severity and clinical laboratory data. We particularly focus on patients with the very common cardiovascular condition, arterial hypertension (AHT), and important factors such as sex, age and medication. Our findings provide a more detailed insight into COVID-19 and which individuals are at higher risk for more severe disease.

Metabolomic profiling of CSF and blood serum elucidates general and sex-specific patterns for mild cognitive impairment and Alzheimer's disease patients.

Background: Beta-amyloid (Abeta) and tau protein in cerebrospinal fluid (CSF) are established diagnostic biomarkers for Alzheimer's disease (AD). However, these biomarkers may not the only ones existing parameters that reflect Alzheimer's disease neuropathological change. The use of quantitative metabolomics approach could provide novel insights into dementia progression and identify key metabolic alterations in CSF and serum. Methods: In the present study, we quantified a set of 45 metabolites in CSF (71 patients) and 27 in serum (76 patients) in patients with mild cognitive impairment (MCI), AD, and controls using nuclear magnetic resonance (NMR)-based metabolomics. Results: We found significantly reduced CSF (1.32-fold, p = 0.0195) and serum (1.47-fold, p = 0.0484) levels of the ketone body acetoacetate in AD and MCI patients. Additionally, we found decreased levels (1.20-fold, p = 0.0438) of the branched-chain amino acid (BCAA) valine in the CSF of AD patients with increased valine degradation pathway metabolites (such as 3-hydroxyisobutyrate and α-ketoisovalerate). Moreover, we discovered that CSF 2-hydroxybutyrate is dramatically reduced in the MCI patient group (1.23-fold, p = 0.039). On the other hand, vitamin C (ascorbate) was significantly raised in CSF of these patients (p = 0.008). We also identified altered CSF protein content, 1,5-anhydrosorbitol and fructose as further metabolic shifts distinguishing AD from MCI. Significantly decreased serum levels of the amino acid ornithine were seen in the AD dementia group when compared to healthy controls (1.36-fold, p = 0.011). When investigating the effect of sex, we found for AD males the sign of decreased 2-hydroxybutyrate and acetoacetate in CSF while for AD females increased serum creatinine was identified. Conclusion: Quantitative NMR metabolomics of CSF and serum was able to efficiently identify metabolic changes associated with dementia groups of MCI and AD patients. Further, we showed strong correlations between these changes and well-established metabolomic and clinical indicators like Abeta.

Pyruvate-conjugation of PEGylated liposomes for targeted drug delivery to retinal photoreceptors.

Despite several promising candidates, there is a paucity of drug treatments available for patients suffering from retinal diseases. An important reason for this is the lack of suitable delivery systems that can achieve sufficiently high drug uptake in the retina and its photoreceptors. A promising and versatile method for drug delivery to specific cell types involves transporter-targeted liposomes, i.e., liposomes surface-coated with substrates for transporter proteins highly expressed on the target cell. We identified strong lactate transporter (monocarboxylate transporter, MCT) expression on photoreceptors as a potential target for drug delivery vehicles. To evaluate MCT suitability for drug targeting, we used PEG-coated liposomes and conjugated these with different monocarboxylates, including lactate, pyruvate, and cysteine. Monocarboxylate-conjugated and dye-loaded liposomes were tested on both human-derived cell-lines and murine retinal explant cultures. We found that liposomes conjugated with pyruvate consistently displayed higher cell uptake than unconjugated liposomes or liposomes conjugated with lactate or cysteine. Pharmacological inhibition of MCT1 and MCT2 reduced internalization, suggesting an MCT-dependent uptake mechanism. Notably, pyruvate-conjugated liposomes loaded with the drug candidate CN04 reduced photoreceptor cell death in the murine rd1 retinal degeneration model while free drug solutions could not achieve the same therapeutic effect. Our study thus highlights pyruvate-conjugated liposomes as a promising system for drug delivery to retinal photoreceptors, as well as other neuronal cell types displaying high expression of MCT-type proteins.

Measuring the Release of Lactate from Wild-Type and rd1 Mouse Retina.

The retina has the highest energy consumption of any tissue in the human body. Remarkably, to satisfy its energy demand, the retina appears to rely mostly on aerobic glycolysis, which results in the production and release of large amounts of lactate. In the present study, we compared two different methods to assess lactate release from in vitro organotypic retinal explants cultured under entirely controlled, serum-free conditions. We used a standard lactate assay kit and 1H-nuclear magnetic resonance (NMR) spectroscopy-based analysis. We found that during the culturing of retinal explants derived from wild-type mice, lactate was released in large amounts and that the two different methods agreed well with each other. When comparing wild-type retina with degenerating rd1 mouse retina, we found the latter to release significantly higher amounts of lactate. Hence, degenerating retina may have an even higher energy demand and metabolic rate compared to healthy retina. We conclude that the use of lactate measurement can be a reliable and simple readout to evaluate ongoing retinal metabolism.

Overexpression of human alpha-Synuclein leads to dysregulated microbiome/metabolites with ageing in a rat model of Parkinson disease.

BACKGROUND: Braak's hypothesis states that sporadic Parkinson's disease (PD) follows a specific progression of pathology from the peripheral to the central nervous system, and this progression can be monitored by detecting the accumulation of alpha-Synuclein (α-Syn) protein. Consequently, there is growing interest in understanding how the gut (commensal) microbiome can regulate α-Syn accumulation, as this could potentially lead to PD. METHODS: We used 16S rRNA and shotgun sequencing to characterise microbial diversity. 1H-NMR was employed to understand the metabolite production and intestinal inflammation estimated using ELISA and RNA-sequencing from feces and the intestinal epithelial layer respectively. The Na+ channel current and gut permeability were measured using an Ussing chamber. Immunohistochemistry and immunofluorescence imaging were applied to detect the α-Syn protein. LC-MS/MS was used for characterization of proteins from metabolite treated neuronal cells. Finally, Metascape and Ingenuity Pathway Analysis (IPA) bioinformatics tools were used for identification of dysregulated pathways. RESULTS: We studied a transgenic (TG) rat model overexpressing the human SNCA gene and found that a progressive gut microbial composition alteration characterized by the reduction of Firmicutes to Bacteroidetes ratio could be detected in the young TG rats. Interestingly, this ratio then increased with ageing. The dynamics of Lactobacillus and Alistipes were monitored and reduced Lactobacillus and increased Alistipes abundance was discerned in ageing TG rats. Additionally, the SNCA gene overexpression resulted in gut α-Syn protein expression and increased with advanced age. Further, older TG animals had increased intestinal inflammation, decreased Na+ current and a robust alteration in metabolite production characterized by the increase of succinate levels in feces and serum. Manipulation of the gut bacteria by short-term antibiotic cocktail treatment revealed a complete loss of short-chain fatty acids and a reduction in succinate levels. Although antibiotic cocktail treatment did not change α-Syn expression in the enteric nervous system of the colon, however, reduced α-Syn expression was detected in the olfactory bulbs (forebrain) of the TG rats. CONCLUSION: Our data emphasize that the gut microbiome dysbiosis synchronous with ageing leads to a specific alteration of gut metabolites and can be modulated by antibiotics which may affect PD pathology.

Maintained imbalance of triglycerides, apolipoproteins, energy metabolites and cytokines in long-term COVID-19 syndrome patients.

Background: Deep metabolomic, proteomic and immunologic phenotyping of patients suffering from an infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have matched a wide diversity of clinical symptoms with potential biomarkers for coronavirus disease 2019 (COVID-19). Several studies have described the role of small as well as complex molecules such as metabolites, cytokines, chemokines and lipoproteins during infection and in recovered patients. In fact, after an acute SARS-CoV-2 viral infection almost 10-20% of patients experience persistent symptoms post 12 weeks of recovery defined as long-term COVID-19 syndrome (LTCS) or long post-acute COVID-19 syndrome (PACS). Emerging evidence revealed that a dysregulated immune system and persisting inflammation could be one of the key drivers of LTCS. However, how these biomolecules altogether govern pathophysiology is largely underexplored. Thus, a clear understanding of how these parameters within an integrated fashion could predict the disease course would help to stratify LTCS patients from acute COVID-19 or recovered patients. This could even allow to elucidation of a potential mechanistic role of these biomolecules during the disease course. Methods: This study comprised subjects with acute COVID-19 (n=7; longitudinal), LTCS (n=33), Recov (n=12), and no history of positive testing (n=73). 1H-NMR-based metabolomics with IVDr standard operating procedures verified and phenotyped all blood samples by quantifying 38 metabolites and 112 lipoprotein properties. Univariate and multivariate statistics identified NMR-based and cytokine changes. Results: Here, we report on an integrated analysis of serum/plasma by NMR spectroscopy and flow cytometry-based cytokines/chemokines quantification in LTCS patients. We identified that in LTCS patients lactate and pyruvate were significantly different from either healthy controls (HC) or acute COVID-19 patients. Subsequently, correlation analysis in LTCS group only among cytokines and amino acids revealed that histidine and glutamine were uniquely attributed mainly with pro-inflammatory cytokines. Of note, triglycerides and several lipoproteins (apolipoproteins Apo-A1 and A2) in LTCS patients demonstrate COVID-19-like alterations compared with HC. Interestingly, LTCS and acute COVID-19 samples were distinguished mostly by their phenylalanine, 3-hydroxybutyrate (3-HB) and glucose concentrations, illustrating an imbalanced energy metabolism. Most of the cytokines and chemokines were present at low levels in LTCS patients compared with HC except for IL-18 chemokine, which tended to be higher in LTCS patients. Conclusion: The identification of these persisting plasma metabolites, lipoprotein and inflammation alterations will help to better stratify LTCS patients from other diseases and could help to predict ongoing severity of LTCS patients.

Stratification of ovarian cancer borderline from high-grade serous carcinoma patients by quantitative serum NMR spectroscopy of metabolites, lipoproteins, and inflammatory markers.

Background: Traditional diagnosis is based on histology or clinical-stage classification which provides no information on tumor metabolism and inflammation, which, however, are both hallmarks of cancer and are directly associated with prognosis and severity. This project was an exploratory approach to profile metabolites, lipoproteins, and inflammation parameters (glycoprotein A and glycoprotein B) of borderline ovarian tumor (BOT) and high-grade serous ovarian cancer (HGSOC) for identifying additional useful serum markers and stratifying ovarian cancer patients in the future. Methods: This project included 201 serum samples of which 50 were received from BOT and 151 from high-grade serous ovarian cancer (HGSOC), respectively. All the serum samples were validated and phenotyped by 1H-NMR-based metabolomics with in vitro diagnostics research (IVDr) standard operating procedures generating quantitative data on 38 metabolites, 112 lipoprotein parameters, and 5 inflammation markers. Uni- and multivariate statistics were applied to identify NMR-based alterations. Moreover, biomarker analysis was carried out with all NMR parameters and CA-125. Results: Ketone bodies, glutamate, 2-hydroxybutyrate, glucose, glycerol, and phenylalanine levels were significantly higher in HGSOC, while the same tumors showed significantly lower levels of alanine and histidine. Furthermore, alanine and histidine and formic acid decreased and increased, respectively, over the clinical stages. Inflammatory markers glycoproteins A and B (GlycA and GlycB) increased significantly over the clinical stages and were higher in HGSOC, alongside significant changes in lipoproteins. Lipoprotein subfractions of VLDLs, IDLs, and LDLs increased significantly in HGSOC and over the clinical stages, while total plasma apolipoprotein A1 and A2 and a subfraction of HDLs decreased significantly over the clinical stages. Additionally, LDL triglycerides significantly increased in advanced ovarian cancer. In biomarker analysis, glycoprotein inflammation biomarkers behaved in the same way as the established clinical biomarker CA-125. Moreover, CA-125/GlycA, CA-125/GlycB, and CA-125/Glycs are potential biomarkers for diagnosis, prognosis, and treatment response of epithelial ovarian cancer (EOC). Last, the quantitative inflammatory parameters clearly displayed unique patterns of metabolites, lipoproteins, and CA-125 in BOT and HGSOC with clinical stages I-IV. Conclusion: 1H-NMR-based metabolomics with commercial IVDr assays could detect and identify altered metabolites and lipoproteins relevant to EOC development and progression and show that inflammation (based on glycoproteins) increased along with malignancy. As inflammation is a hallmark of cancer, glycoproteins, thereof, are promising future serum biomarkers for the diagnosis, prognosis, and treatment response of EOC. This was supported by the definition and stratification of three different inflammatory serum classes which characterize specific alternations in metabolites, lipoproteins, and CA-125, implicating that future diagnosis could be refined not only by diagnosed histology and/or clinical stages but also by glycoprotein classes.

Repurposing the mucolytic agent ambroxol for treatment of sub-acute and chronic ischaemic stroke.

Ambroxol is a well-known mucolytic expectorant, which has gained much attention in amyotrophic lateral sclerosis, Parkinson's and Gaucher's disease. A specific focus has been placed on ambroxol's glucocerebrosidase-stimulating activity, on grounds that the point mutation of the gba1 gene, which codes for this enzyme, is a risk factor for developing Parkinson's disease. However, ambroxol has been attributed other characteristics, such as the potent inhibition of sodium channels, modification of calcium homeostasis, anti-inflammatory effects and modifications of oxygen radical scavengers. We hypothesized that ambroxol could have a direct impact on neuronal rescue if administered directly after ischaemic stroke induction. We longitudinally evaluated 53 rats using magnetic resonance imaging to examine stroke volume, oedema, white matter integrity, resting state functional MRI and behaviour for 1 month after ischemic stroke onset. For closer mechanistic insights, we evaluated tissue metabolomics of different brain regions in a subgroup of animals using ex vivo nuclear magnetic resonance spectroscopy. Ambroxol-treated animals presented reduced stroke volumes, reduced cytotoxic oedema, reduced white matter degeneration, reduced necrosis, improved behavioural outcomes and complex changes in functional brain connectivity. Nuclear magnetic resonance spectroscopy tissue metabolomic data at 24 h post-stroke proposes several metabolites that are capable of minimizing post-ischaemic damage and that presented prominent shifts during ambroxol treatment in comparison to controls. Taking everything together, we propose that ambroxol catalyzes recovery in energy metabolism, cellular homeostasis, membrane repair mechanisms and redox balance. One week of ambroxol administration following stroke onset reduced ischaemic stroke severity and improved functional outcome in the subacute phase followed by reduced necrosis in the chronic stroke phase.

TGF-Beta Modulates the Integrity of the Blood Brain Barrier In Vitro, and Is Associated with Metabolic Alterations in Pericytes.

The blood-brain barrier (BBB) is a selectively permeable boundary that separates the circulating blood from the extracellular fluid of the brain and is an essential component for brain homeostasis. In glioblastoma (GBM), the BBB of peritumoral vessels is often disrupted. Pericytes, being important to maintaining BBB integrity, can be functionally modified by GBM cells which induce proliferation and cell motility via the TGF-ß-mediated induction of central epithelial to mesenchymal transition (EMT) factors. We demonstrate that pericytes strengthen the integrity of the BBB in primary endothelial cell/pericyte co-cultures as an in vitro BBB model, using TEER measurement of the barrier integrity. In contrast, this effect was abrogated by TGF-ß or conditioned medium from TGF-ß secreting GBM cells, leading to the disruption of a so far intact and tight BBB. TGF-ß notably changed the metabolic behavior of pericytes, by shutting down the TCA cycle, driving energy generation from oxidative phosphorylation towards glycolysis, and by modulating pathways that are necessary for the biosynthesis of molecules used for proliferation and cell division. Combined metabolomic and transcriptomic analyses further underscored that the observed functional and metabolic changes of TGF-ß-treated pericytes are closely connected with their role as important supporting cells during angiogenic processes.

MR-spectroscopy in metachromatic leukodystrophy: A model free approach and clinical correlation.

BACKGROUND AND PURPOSE: Metachromatic leukodystrophy (MLD) is a lysosomal enzyme deficiency disorder leading to demyelination and subsequently to a progressive decline in cognitive and motor function. It affects mainly white matter where changes during the course of the disease can be visualized on T2-weighted MRI as hyperintense areas. Associated changes in brain metabolism can be quantified by MR spectroscopy (MRS) and may give complementary information as biomarkers for disease characterisation and progression. Our study aimed to further investigate the correlation of MRS with clinical parameters for motor and cognitive function by using a model free MRS analysis approach that would be precise and straightforward to implement. MATERIALS AND METHODS: 53 MRS datasets derived from 29 patients (10 late-infantile, 19 juvenile) and 12 controls were acquired using a semi-LASER CSI sequence covering a slice through the centrum semiovale above the corpus callosum. We defined four regions of interest in the white matter (frontal white matter [FWM] and the cortico-spinal tract [CST] area, each left and right) and one in cortical grey matter. Spectra were analysed using a model and fitting free approach by calculating the definite integral of 10 intervals which were distributed along the whole spectrum. These 10 intervals were orientated towards the main peaks of the metabolites N-acetylaspartate (NAA), creatine, myo-inositol, choline, glutamine/glutamate and aspartate to approximately attribute changes in the intervals to corresponding metabolites. Their ratios to the main creatine peak integral were correlated with clinical parameters assessing motor and cognitive abilities. Furthermore, in a post-hoc analysis, NAA levels of a subset of 21 MR datasets were correlated to NAA levels in urine measured by 1H (proton) nuclear magnetic resonance (NMR) spectroscopy. The applied interval integration method was validated in the control cohort against the standard approach, using spectral profile templates of known metabolites (LCModel). Both methods showed good agreement, with coefficients of variance being slightly lower for our approach compared to the related LCModel results. Moreover, the new approach was able to extract information out of the frequency range around the main peaks of aspartate and glutamine where LCModel showed only few usable values for the respective metabolites. RESULTS: MLD spectra clearly differed from controls. The most pronounced differences were found in white matter (much less in grey matter), with larger values corresponding to main peaks of myo-inositol, choline and aspartate, and smaller values associated with NAA and glutamine. Late-infantile patients had more severe changes compared to later-onset patients, especially in intervals corresponding to NAA, aspartate, myo-inositol, choline and glutamine. There was a high correlation of several intervals in the corticospinal tract region with motor function (with the most relevant interval corresponding to NAA peak with a correlation coefficient of -0.75; p < 0.001), while cognitive function, by means of IQ, was found to be most correlating in frontal white matter corresponding to the NAA peak (r = 0.84, p < 0.001). The post-hoc analysis showed that the main NAA peak interval correlated negatively with the NAA in urine (r = -0.584, p < 0.001). CONCLUSION: The applied model and fitting free interval integration approach to analyse MRS data of a semi-LASER sequence at 3T suits well to detect and quantify pathological changes in MLD patients through the different courses of the disease and correlates well with clinical symptoms while showing smaller dimensions of variation compared to the more sophisticated single metabolite analysis using LCModel. NAA seems the most clinically meaningful biomarker to use in this context. Its correlation with urine measurements further underlines its potential as a clinically and biologically useful parameter of disease progression in MLD.