Publisher Correction: The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

An Amendment to this paper has been published and can be accessed via a link at the top of the paper.

The PIDDosome activates p53 in response to supernumerary centrosomes.

Centrosomes, the main microtubule-organizing centers in animal cells, are replicated exactly once during the cell division cycle to form the poles of the mitotic spindle. Supernumerary centrosomes can lead to aberrant cell division and have been causally linked to chromosomal instability and cancer. Here, we report that an increase in the number of mature centrosomes, generated by disrupting cytokinesis or forcing centrosome overduplication, triggers the activation of the PIDDosome multiprotein complex, leading to Caspase-2-mediated MDM2 cleavage, p53 stabilization, and p21-dependent cell cycle arrest. This pathway also restrains the extent of developmentally scheduled polyploidization by regulating p53 levels in hepatocytes during liver organogenesis. Taken together, the PIDDosome acts as a first barrier, engaging p53 to halt the proliferation of cells carrying more than one mature centrosome to maintain genome integrity.

Dopamine­iron homeostasis interaction rescues mitochondrial fitness in Parkinson's disease.

Imbalances of iron and dopamine metabolism along with mitochondrial dysfunction have been linked to the pathogenesis of Parkinson's disease (PD). We have previously suggested a direct link between iron homeostasis and dopamine metabolism, as dopamine can increase cellular uptake of iron into macrophages thereby promoting oxidative stress responses. In this study, we investigated the interplay between iron, dopamine, and mitochondrial activity in neuroblastoma SH-SY5Y cells and human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons differentiated from a healthy control and a PD patient with a mutation in the α-synuclein (SNCA) gene. In SH-SY5Y cells, dopamine treatment resulted in increased expression of the transmembrane iron transporters transferrin receptor 1 (TFR1), ferroportin (FPN), and mitoferrin2 (MFRN2) and intracellular iron accumulation, suggesting that dopamine may promote iron uptake. Furthermore, dopamine supplementation led to reduced mitochondrial fitness including decreased mitochondrial respiration, increased cytochrome c control efficiency, reduced mtDNA copy number and citrate synthase activity, increased oxidative stress and impaired aconitase activity. In dopaminergic neurons derived from a healthy control individual, dopamine showed comparable effects as observed in SH-SY5Y cells. The hiPSC-derived PD neurons harboring an endogenous SNCA mutation demonstrated altered mitochondrial iron homeostasis, reduced mitochondrial capacity along with increased oxidative stress and alterations of tricarboxylic acid cycle linked metabolic pathways compared with control neurons. Importantly, dopamine treatment of PD neurons promoted a rescue effect by increasing mitochondrial respiration, activating antioxidant stress response, and normalizing altered metabolite levels linked to mitochondrial function. These observations provide evidence that dopamine affects iron homeostasis, intracellular stress responses and mitochondrial function in healthy cells, while dopamine supplementation can restore the disturbed regulatory network in PD cells.

The metabolite BH4 controls T cell proliferation in autoimmunity and cancer.

Genetic regulators and environmental stimuli modulate T cell activation in autoimmunity and cancer. The enzyme co-factor tetrahydrobiopterin (BH4) is involved in the production of monoamine neurotransmitters, the generation of nitric oxide, and pain1,2. Here we uncover a link between these processes, identifying a fundamental role for BH4 in T cell biology. We find that genetic inactivation of GTP cyclohydrolase 1 (GCH1, the rate-limiting enzyme in the synthesis of BH4) and inhibition of sepiapterin reductase (the terminal enzyme in the synthetic pathway for BH4) severely impair the proliferation of mature mouse and human T cells. BH4 production in activated T cells is linked to alterations in iron metabolism and mitochondrial bioenergetics. In vivo blockade of BH4 synthesis abrogates T-cell-mediated autoimmunity and allergic inflammation, and enhancing BH4 levels through GCH1 overexpression augments responses by CD4- and CD8-expressing T cells, increasing their antitumour activity in vivo. Administration of BH4 to mice markedly reduces tumour growth and expands the population of intratumoral effector T cells. Kynurenine-a tryptophan metabolite that blocks antitumour immunity-inhibits T cell proliferation in a manner that can be rescued by BH4. Finally, we report the development of a potent SPR antagonist for possible clinical use. Our data uncover GCH1, SPR and their downstream metabolite BH4 as critical regulators of T cell biology that can be readily manipulated to either block autoimmunity or enhance anticancer immunity.

Alterations of blood monocyte subset distribution and surface phenotype are linked to infection severity in COVID-19 inpatients.

Severe coronavirus disease 19 (COVID-19) manifests with systemic immediate proinflammatory innate immune activation and altered iron turnover. Iron homeostasis, differentiation, and function of myeloid leukocytes are interconnected. Therefore, we characterized the cellularity, surface marker expression, and iron transporter phenotype of neutrophils and monocyte subsets in COVID-19 patients within 72 h from hospital admission, and analyzed how these parameters relate to infection severity. Between March and November 2020, blood leukocyte samples from hospitalized COVID-19 patients (n = 48) and healthy individuals (n = 7) were analyzed by flow cytometry enabling comparative analysis of 40 features. Inflammation-driven neutrophil expansion, depletion of CD16+ nonclassical monocytes, and changes in surface expression of neutrophil and monocyte CD64 and CD86 were associated with COVID-19 severity. By unsupervised self-organizing map clustering, four patterns of innate myeloid response were identified and linked to varying levels of systemic inflammation, altered cellular iron trafficking and the severity of disease. These alterations of the myeloid leukocyte compartment during acute COVID-19 may be hallmarks of inefficient viral control and immune hyperactivation and may help at risk prediction and treatment optimization.

Comparative analysis of oral and intravenous iron therapy in rat models of inflammatory anemia and iron deficiency.

Anemia is a major health issue and associated with increased morbidity. Iron deficiency anemia (IDA) is the most prevalent, followed by anemia of chronic disease (ACD). IDA and ACD often co-exist, challenging diagnosis and treatment. While iron supplementation is the first-line therapy for IDA, its optimal route of administration and the efficacy of different repletion strategies in ACD are elusive. Female Lewis rats were injected with group A streptococcal peptidoglycan-polysaccharide (PG-APS) to induce inflammatory arthritis with associated ACD and/or repeatedly phlebotomized and fed with a low iron diet to induce IDA, or a combination thereof (ACD/IDA). Iron was either supplemented by daily oral gavage of ferric maltol or by weekly intravenous (i.v.) injection of ferric carboxymaltose for up to 4 weeks. While both strategies reversed IDA, they remained ineffective to improve hemoglobin (Hb) levels in ACD, although oral iron showed slight amelioration of various erythropoiesis-associated parameters. In contrast, both iron treatments significantly increased Hb in ACD/IDA. In ACD and ACD/IDA animals, i.v. iron administration resulted in iron trapping in liver and splenic macrophages, induction of ferritin expression and increased circulating levels of the iron hormone hepcidin and the inflammatory cytokine interleukin-6, while oral iron supplementation reduced interleukin-6 levels. Thus, oral and i.v. iron resulted in divergent effects on systemic and tissue iron homeostasis and inflammation. Our results indicate that both iron supplements improve Hb in ACD/IDA, but are ineffective in ACD with pronounced inflammation, and that under the latter condition, i.v. iron is trapped in macrophages and may enhance inflammation.

Phenotyping of Acute and Persistent Coronavirus Disease 2019 Features in the Outpatient Setting: Exploratory Analysis of an International Cross-sectional Online Survey.

BACKGROUND: Long COVID, defined as the presence of coronavirus disease 2019 (COVID-19) symptoms ≥28 days after clinical onset, is an emerging challenge to healthcare systems. The objective of the current study was to explore recovery phenotypes in nonhospitalized individuals with COVID-19. METHODS: A dual cohort, online survey study was conducted between September 2020 and July 2021 in the neighboring European regions Tyrol (TY; Austria, n = 1157) and South Tyrol (STY; Italy, n = 893). Data were collected on demographics, comorbid conditions, COVID-19 symptoms, and recovery in adult outpatients. Phenotypes of acute COVID-19, postacute sequelae, and risk of protracted recovery were explored using semi-supervised clustering and multiparameter least absolute shrinkage and selection operator (LASSO) modeling. RESULTS: Participants in the study cohorts were predominantly working age (median age [interquartile range], 43 [31-53] years] for TY and 45 [35-55] years] for STY) and female (65.1% in TY and 68.3% in STY). Nearly half (47.6% in TY and 49.3% in STY) reported symptom persistence beyond 28 days. Two acute COVID-19 phenotypes were discerned: the nonspecific infection phenotype and the multiorgan phenotype (MOP). Acute MOP symptoms encompassing multiple neurological, cardiopulmonary, gastrointestinal, and dermatological symptoms were linked to elevated risk of protracted recovery. The major subset of individuals with long COVID (49.3% in TY; 55.6% in STY) displayed no persistent hyposmia or hypogeusia but high counts of postacute MOP symptoms and poor self-reported physical recovery. CONCLUSIONS: The results of our 2-cohort analysis delineated phenotypic diversity of acute and postacute COVID-19 manifestations in home-isolated patients, which must be considered in predicting protracted convalescence and allocating medical resources.

A fully human anti-BMP6 antibody reduces the need for erythropoietin in rodent models of the anemia of chronic disease.

Recombinant erythropoietin (EPO) and iron substitution are a standard of care for treatment of anemias associated with chronic inflammation, including anemia of chronic kidney disease. A black box warning for EPO therapy and concerns about negative side effects related to high-dose iron supplementation as well as the significant proportion of patients becoming EPO resistant over time explains the medical need to define novel strategies to ameliorate anemia of chronic disease (ACD). As hepcidin is central to the iron-restrictive phenotype in ACD, therapeutic approaches targeting hepcidin were recently developed. We herein report the therapeutic effects of a fully human anti-BMP6 antibody (KY1070) either as monotherapy or in combination with Darbepoetin alfa on iron metabolism and anemia resolution in 2 different, well-established, and clinically relevant rodent models of ACD. In addition to counteracting hepcidin-driven iron limitation for erythropoiesis, we found that the combination of KY1070 and recombinant human EPO improved the erythroid response compared with either monotherapy in a qualitative and quantitative manner. Consequently, the combination of KY1070 and Darbepoetin alfa resulted in an EPO-sparing effect. Moreover, we found that suppression of hepcidin via KY1070 modulates ferroportin expression on erythroid precursor cells, thereby lowering potentially toxic-free intracellular iron levels and by accelerating erythroid output as reflected by increased maturation of erythrocyte progenitors. In summary, we conclude that treatment of ACD, as a highly complex disease, becomes more effective by a multifactorial therapeutic approach upon mobilization of endogenous iron deposits and stimulation of erythropoiesis.

A spark of hope: histopathological and functional recovery after critical COVID-19.

BACKGROUND: There are substantial concerns about fibrotic and vascular pulmonary sequelae after coronavirus disease 2019 (COVID-19) associated acute respiratory distress syndrome (ARDS).AQ1 Histopathology reports of lung biopsies from COVID-19 survivors are scarce. CASE: We herein report results of functional and histopathological studies in a 70 year-old man undergoing a co-incidental tumor lobectomy six months after long-term mechanical ventilation for COVID-19 pneumonia. CONCLUSION: Despite several unfavorable risk factors, this case presentation shows a completed pulmonary recovery process within a few months.

Anemia of inflammation.

Anemia of inflammation (AI), also known as anemia of chronic disease (ACD), is regarded as the most frequent anemia in hospitalized and chronically ill patients. It is prevalent in patients with diseases that cause prolonged immune activation, including infection, autoimmune diseases, and cancer. More recently, the list has grown to include chronic kidney disease, congestive heart failure, chronic pulmonary diseases, and obesity. Inflammation-inducible cytokines and the master regulator of iron homeostasis, hepcidin, block intestinal iron absorption and cause iron retention in reticuloendothelial cells, resulting in iron-restricted erythropoiesis. In addition, shortened erythrocyte half-life, suppressed erythropoietin response to anemia, and inhibition of erythroid cell differentiation by inflammatory mediators further contribute to AI in a disease-specific pattern. Although the diagnosis of AI is a diagnosis of exclusion and is supported by characteristic alterations in iron homeostasis, hypoferremia, and hyperferritinemia, the diagnosis of AI patients with coexisting iron deficiency is more difficult. In addition to treatment of the disease underlying AI, the combination of iron therapy and erythropoiesis-stimulating agents can improve anemia in many patients. In the future, emerging therapeutics that antagonize hepcidin function and redistribute endogenous iron for erythropoiesis may offer additional options. However, based on experience with anemia treatment in chronic kidney disease, critical illness, and cancer, finding the appropriate indications for the specific treatment of AI will require improved understanding and a balanced consideration of the contribution of anemia to each patient's morbidity and the impact of anemia treatment on the patient's prognosis in a variety of disease settings.

Leishmania donovani Exploits Macrophage Heme Oxygenase-1 To Neutralize Oxidative Burst and TLR Signaling-Dependent Host Defense.

Suppression of host oxidative burst is essential for survival of the intracellular parasite Leishmania donovani Screening of macrophage antioxidant enzymes during infection revealed marked upregulation of the heme-degrading enzyme, heme oxygenase-1 (HO-1). Moreover, HO-1-silenced RAW macrophages depicted increased superoxide production and decreased parasite survival. HO-1 induction decreased cellular heme content, thereby inhibiting the heme-dependent maturation of gp91phox, a catalytic component of major reactive oxygen species-producing enzyme NAD(P)H oxidase. Decreased gp91phox expression resulted in reduced stability of p22phox, another component of the catalytic center of NAD(P)H oxidase. Replenishing infected cells with exogenous heme reversed these effects and restored NAD(P)H oxidase activity. Persistent HO-1 expression at late hour of infection prompted us to investigate its effect on other host defense parameters, and inhibition study revealed a reciprocal relationship of HO-1 with host proinflammatory responses. Among all the HO-1-mediated heme degradation products (CO, Fe, and biliverdin), only CO documented potent anti-inflammatory effects. Quenching of CO during infection increased the production of disease-resolving cytokines IL-12 and TNF-α. Coimmunoprecipitation experiments revealed that CO inhibited the interaction of TLR4 with MyD88 and TIR domain-containing adapter-inducing IFN-ß, thereby dampening the activation of NF-κB and IFN regulatory factor 3-mediated production of proinflammatory cytokines. Administration of HO-1 inhibitor tin protoporphyrin IX dichloride in infected BALB/c mice led to a decrease in liver and spleen parasite burden along with increased production of IL-12 and TNF-α. These results suggest that HO-1 on one hand inhibits reactive oxygen species generation and on the other hand downregulates host favorable cytokine responses, thereby facilitating intramacrophage parasite survival.

Assessing global COPD awareness with Google Trends.

Although chronic obstructive pulmonary disease (COPD) prevalence and mortality rates rise continuously, patients often remain undiagnosed, probably due to a lack of disease-related awareness. The aim of this study was to quantify public interest in COPD by analysing the frequency of web queries via Google.Data from 2004 to 2018 were collected using the search engine query data analysis tool Google Trends. The relative search volume of the topic "chronic obstructive pulmonary disease" was compared with the relative search volume of nine topics representing the major causes of death in high-income countries according to the World Health Organization.Our analysis showed highest relative search volumes for the topics "diabetes mellitus", followed by "stroke" and "breast cancer". The topic "chronic obstructive pulmonary disease" ranked eighth and its relative search volume clearly displayed a seasonal variation, with peaks in the first and the fourth quarter of the year.This analysis reveals that COPD is highly under-represented in the public interest, while real-world prevalence constantly rises, indicating that there is still an urgent need to raise the levels of awareness for COPD.

Momelotinib inhibits ACVR1/ALK2, decreases hepcidin production, and ameliorates anemia of chronic disease in rodents.

Patients with myelofibrosis (MF) often develop anemia and frequently become dependent on red blood cell transfusions. Results from a phase 2 study for the treatment of MF with the Janus kinase 1/2 (JAK1/2) inhibitor momelotinib (MMB) demonstrated that MMB treatment ameliorated anemia, which was unexpected for a JAK1/2 inhibitor, because erythropoietin-mediated JAK2 signaling is essential for erythropoiesis. Using a rat model of anemia of chronic disease, we demonstrated that MMB treatment can normalize hemoglobin and red blood cell numbers. We found that this positive effect is driven by direct inhibition of the bone morphogenic protein receptor kinase activin A receptor, type I (ACVR1), and the subsequent reduction of hepatocyte hepcidin production. Of note, ruxolitinib, a JAK1/2 inhibitor approved for the treatment of MF, had no inhibitory activity on this pathway. Further, we demonstrated the effect of MMB is not mediated by direct inhibition of JAK2-mediated ferroportin (FPN1) degradation, because neither MMB treatment nor myeloid-specific deletion of JAK2 affected FPN1 expression. Our data support the hypothesis that the improvement of inflammatory anemia by MMB results from inhibition of ACVR1-mediated hepcidin expression in the liver, which leads to increased mobilization of sequestered iron from cellular stores and subsequent stimulation of erythropoiesis.

Association of mitochondrial iron deficiency and dysfunction with idiopathic restless legs syndrome.

BACKGROUND: Restless legs syndrome is a sensorimotor neurological disorder of the limbs that impairs quality of life and disturbs sleep. However, there has been progress in understanding the disease involving the dopaminergic system as well as iron metabolism. The exact pathophysiological mechanisms of restless legs syndrome remain elusive. We tried to elucidate the underlying mechanisms in iron metabolism in restless legs syndrome subjects on a systemic, cellular, and mitochondrial level. METHODS: We conducted a study prospectively recruiting 168 restless legs syndrome patients and 119 age-matched healthy controls focusing on iron metabolism using human monocytes as surrogates. RESULTS: Evaluation of systemic iron metabolism parameters in the circulation showed no significant difference between patients and controls. We observed a significant reduction in mRNA levels of heme oxygenase 1 and mitochondrial iron genes like mitoferrin 1 and 2 in monocytes isolated from restless legs syndrome patients, indicating mitochondrial iron deficiency. Interestingly, we also observed reduced expression of iron regulatory protein 2 along with impaired activity of mitochondrial aconitase and reduced mitochondrial superoxide formation in restless legs syndrome subjects. Along this line, patients had reduced mitochondrial respiratory capacity that improved in restless legs syndrome subjects under treatment with dopaminergic drugs compared with untreated patients. CONCLUSIONS: Our data suggest that restless legs syndrome is linked to mitochondrial iron deficiency and associated impairment of mitochondrial function. This is partly corrected by treatment with dopaminergic drugs compared with untreated patients, which may be linked to an effect of dopamine on cellular iron homeostasis. © 2018 International Parkinson and Movement Disorder Society.

Cystic echinococcosis in the thigh: a case report.

Intramuscular cystic echinococcosis is a very rare occurrence. Herein we report a case of a 37-year-old patient who presented with progressive swelling of his left thigh. Ultrasound evaluation showed a multicystic, encapsulated lesion (16 × 3.5 × 8.5 cm) in the M. vastus lateralis, and serology confirmed the diagnosis of Echinococcus granulosus s.l. infection. No additional cysts were detected upon total body CT scan. The patient was treated with albendazole pre-operatively; surgical resection of the mass was then successfully performed. The patient feels well and no signs of residual infestation were seen after 2 years of follow-up.

Salmonella Utilizes Zinc To Subvert Antimicrobial Host Defense of Macrophages via Modulation of NF-κB Signaling.

Zinc sequestration by macrophages is considered a crucial host defense strategy against infection by the intracellular bacterium Salmonella enterica serovar Typhimurium. However, the underlying mechanisms remain elusive. In this study, we found that zinc favors pathogen survival within macrophages. Salmonella-hosting macrophages contained higher free zinc levels than did uninfected macrophages and cells that successfully eliminated bacteria, which was paralleled by the impaired production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in bacterium-harboring cells. A profound, zinc-mediated inhibition of NF-κB p65 transcriptional activity affecting the expression of the ROS- and RNS-forming enzymes phos47 and inducible nitric oxide synthase (iNOS) provided a mechanistic explanation for this phenomenon. Macrophages responded to infection by enhancing the expression of zinc-scavenging metallothioneins 1 and 2, whose genetic deletion caused increased free zinc levels, reduced ROS and RNS production, and increased the survival of Salmonella Our data suggest that Salmonella invasion of macrophages results in a bacterium-driven increase in the intracellular zinc level, which weakens antimicrobial defense and the ability of macrophages to eradicate the pathogen. Thus, limitation of cytoplasmic zinc levels may help to control infection by intracellular bacteria.

"Pumping iron"-how macrophages handle iron at the systemic, microenvironmental, and cellular levels.

Macrophages reside in virtually every organ. First arising during embryogenesis, macrophages replenish themselves in the adult through a combination of self-renewal and influx of bone marrow-derived monocytes. As large phagocytic cells, macrophages participate in innate immunity while contributing to tissue-specific homeostatic functions. Among the key metabolic tasks are senescent red blood cell recycling, free heme detoxification, and provision of iron for de novo hemoglobin synthesis. While this systemic mechanism involves the shuttling of iron between spleen, liver, and bone marrow through the concerted function of defined macrophage populations, similar circuits appear to exist within the microenvironment of other organs. The high turnover of iron is the prerequisite for continuous erythropoiesis and tissue integrity but challenges macrophages' ability to maintain cellular iron homeostasis and immune function.This review provides a brief overview of systemic, microenvironmental, and cellular aspects of macrophage iron handling with a focus on exciting and unresolved questions in the field.

Heme oxygenase 1 controls early innate immune response of macrophages to Salmonella Typhimurium infection.

Macrophages are central for the immune control of intracellular microbes. Heme oxygenase 1 (HO-1, hmox) is the first and rate limiting enzyme in the breakdown of heme originating from degraded senescent erythrocytes and heme-proteins, yielding equal amounts of iron, carbon monoxide and biliverdin. HO-1 is strongly up-regulated in macrophages in response to inflammatory signals, including bacterial endotoxin. In view of the essential role of iron for the growth and proliferation of intracellular bacteria along with known effects of the metal on innate immune function, we examined whether HO-1 plays a role in the control of infection with the intracellular bacterium Salmonella Typhimurium. We studied the course of infection in stably-transfected murine macrophages (RAW264.7) bearing a tetracycline-inducible plasmid producing hmox shRNA and in primary HO-1 knockout macrophages. While uptake of bacteria into macrophages was not affected, a significantly reduced survival of intracellular Salmonella was observed upon hmox knockdown or pharmacological hmox inhibition, which was independent of Nramp1 functionality. This could be traced to limitation of iron availability for intramacrophage bacteria along with enhanced stimulation of innate immune effector pathways, including the formation of reactive oxygen and nitrogen species and increased TNF-α expression. Mechanistically, these latter effects result from intracellular iron limitation with subsequent activation of NF-κB and further inos, tnfa and p47phox transcription along with reduced formation of the anti-inflammatory and radical scavenging molecules, CO and biliverdin as a consequence of HO-1 silencing. Taken together our data provide novel evidence that the infection-driven induction of HO-1 exerts detrimental effects in the early control of Salmonella infection, whereas hmox inhibition can favourably modulate anti-bacterial immune effector pathways of macrophages and promote bacterial elimination.

Pre-analytic evaluation of volumetric absorptive microsampling and integration in a mass spectrometry-based metabolomics workflow.

Volumetric absorptive microsampling (VAMS) is a novel approach that allows single-drop (10 µL) blood collection. Integration of VAMS with mass spectrometry (MS)-based untargeted metabolomics is an attractive solution for both human and animal studies. However, to boost the use of VAMS in metabolomics, key pre-analytical questions need to be addressed. Therefore, in this work, we integrated VAMS in a MS-based untargeted metabolomics workflow and investigated pre-analytical strategies such as sample extraction procedures and metabolome stability at different storage conditions. We first evaluated the best extraction procedure for the polar metabolome and found that the highest number and amount of metabolites were recovered upon extraction with acetonitrile/water (70:30). In contrast, basic conditions (pH 9) resulted in divergent metabolite profiles mainly resulting from the extraction of intracellular metabolites originating from red blood cells. In addition, the prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but once the VAMS devices were stored at - 80 °C, the metabolome remained stable for up to 6 months. The time used for drying the sample did also affect the metabolome. In fact, some metabolites were rapidly degraded or accumulated in the sample during the first 48 h at room temperature, indicating that a longer drying step will significantly change the concentration in the sample. Graphical abstract Volumetric absorptive microsampling (VAMS) is a novel technology that allows single-drop blood collection and, in combination with mass spectrometry (MS)-based untargeted metabolomics, represents an attractive solution for both human and animal studies. In this work, we integrated VAMS in a MS-based untargeted metabolomics workflow and investigated pre-analytical strategies such as sample extraction procedures and metabolome stability at different storage conditions. The latter revealed that prolonged storage of blood samples at room temperature caused significant changes in metabolome composition, but if VAMS devices were stored at - 80 °C, the metabolome remained stable for up to 6 months.