COVID-19: A complex disease with a unique metabolic signature.

Plasma of COVID-19 patients contains a strong metabolomic/lipoproteomic signature, revealed by the NMR analysis of a cohort of >500 patients sampled during various waves of COVID-19 infection, corresponding to the spread of different variants, and having different vaccination status. This composite signature highlights common traits of the SARS-CoV-2 infection. The most dysregulated molecules display concentration trends that scale with disease severity and might serve as prognostic markers for fatal events. Metabolomics evidence is then used as input data for a sex-specific multi-organ metabolic model. This reconstruction provides a comprehensive view of the impact of COVID-19 on the entire human metabolism. The human (male and female) metabolic network is strongly impacted by the disease to an extent dictated by its severity. A marked metabolic reprogramming at the level of many organs indicates an increase in the generic energetic demand of the organism following infection. Sex-specific modulation of immune response is also suggested.

Profiling metabolites and lipoproteins in COMETA, an Italian cohort of COVID-19 patients.

Metabolomics and lipidomics have been used in several studies to define the biochemical alterations induced by COVID-19 in comparison with healthy controls. Those studies highlighted the presence of a strong signature, attributable to both metabolites and lipoproteins/lipids. Here, 1H NMR spectra were acquired on EDTA-plasma from three groups of subjects: i) hospitalized COVID-19 positive patients (≤21 days from the first positive nasopharyngeal swab); ii) hospitalized COVID-19 positive patients (>21 days from the first positive nasopharyngeal swab); iii) subjects after 2-6 months from SARS-CoV-2 eradication. A Random Forest model built using the EDTA-plasma spectra of COVID-19 patients ≤21 days and Post COVID-19 subjects, provided a high discrimination accuracy (93.6%), indicating both the presence of a strong fingerprint of the acute infection and the substantial metabolic healing of Post COVID-19 subjects. The differences originate from significant alterations in the concentrations of 16 metabolites and 74 lipoprotein components. The model was then used to predict the spectra of COVID-19>21 days subjects. In this group, the metabolite levels are closer to those of the Post COVID-19 subjects than to those of the COVID-19≤21 days; the opposite occurs for the lipoproteins. Within the acute phase patients, characteristic trends in metabolite levels are observed as a function of the disease severity. The metabolites found altered in COVID-19≤21 days patients with respect to Post COVID-19 individuals overlap with acute infection biomarkers identified previously in comparison with healthy subjects. Along the trajectory towards healing, the metabolome reverts back to the "healthy" state faster than the lipoproteome.

Unlocking the Power of Human Ferritin: Enhanced Drug Delivery of Aurothiomalate in A2780 Ovarian Cancer Cells.

Aurothiomalate (AuTM) is an FDA-approved antiarthritic gold drug with unique anticancer properties. To enhance its anticancer activity, we prepared a bioconjugate with human apoferritin (HuHf) by attaching some AuTM moieties to surface protein residues. The reaction of apoferritin with excess AuTM yielded a single adduct, that was characterized by ESI MS and ICP-OES analysis, using three mutant ferritins and trypsinization experiments. The adduct contains ~3 gold atoms per ferritin subunit, arranged in a small cluster bound to Cys90 and Cys102. MD simulations provide a plausible structural model for the cluster. The adduct was evaluated for its pharmacological properties and was found to be significantly more cytotoxic than free AuTM against A2780 cancer cells mainly due to higher gold uptake. NMR-metabolomics showed that AuTM bound to HuHf and free AuTM induced qualitatively similar changes in treated cancer cells, indicating that the effects on cell metabolism are approximately the same, in agreement with independent biochemical experiments. In conclusion, we have demonstrated here that a molecularly precise bioconjugate formed between AuTM and HuHf exhibits anticancer properties far superior to the free drug, while retaining its key mechanistic features. Evidence is provided that human ferritin can serve as an excellent carrier for this metallodrug.

Metabolomic/lipidomic profiling of COVID-19 and individual response to tocilizumab.

The current pandemic emergence of novel coronavirus disease (COVID-19) poses a relevant threat to global health. SARS-CoV-2 infection is characterized by a wide range of clinical manifestations, ranging from absence of symptoms to severe forms that need intensive care treatment. Here, plasma-EDTA samples of 30 patients compared with age- and sex-matched controls were analyzed via untargeted nuclear magnetic resonance (NMR)-based metabolomics and lipidomics. With the same approach, the effect of tocilizumab administration was evaluated in a subset of patients. Despite the heterogeneity of the clinical symptoms, COVID-19 patients are characterized by common plasma metabolomic and lipidomic signatures (91.7% and 87.5% accuracy, respectively, when compared to controls). Tocilizumab treatment resulted in at least partial reversion of the metabolic alterations due to SARS-CoV-2 infection. In conclusion, NMR-based metabolomic and lipidomic profiling provides novel insights into the pathophysiological mechanism of human response to SARS-CoV-2 infection and to monitor treatment outcomes.

NMR-Based Metabolomics to Evaluate Individual Response to Treatments.

The aim of this chapter is to highlight the various aspects of metabolomics in relation to health and diseases, starting from the definition of metabolic space and of how individuals tend to maintain their own position in this space. Physio-pathological stimuli may cause individuals to lose their position and then regain it, or move irreversibly to other positions. By way of examples, mostly selected from our own work using 1H NMR on biological fluids, we describe the effects on the individual metabolomic fingerprint of mild external interventions, such as diet or probiotic administration. Then we move to pathologies (such as celiac disease, various types of cancer, viral infections, and other diseases), each characterized by a well-defined metabolomic fingerprint. We describe the effects of drugs on the disease fingerprint and on its reversal to a healthy metabolomic status. Drug toxicity can be also monitored by metabolomics. We also show how the individual metabolomic fingerprint at the onset of a disease may discriminate responders from non-responders to a given drug, or how it may be prognostic of e.g., cancer recurrence after many years. In parallel with fingerprinting, profiling (i.e., the identification and quantification of many metabolites and, in the case of selected biofluids, of the lipoprotein components that contribute to the 1H NMR spectral features) can provide hints on the metabolic pathways that are altered by a disease and assess their restoration after treatment.

Serum or Plasma (and Which Plasma), That Is the Question.

Blood derivatives are the biofluids of choice for metabolomic clinical studies since blood can be collected with low invasiveness and is rich in biological information. However, the choice of the blood collection tubes has an undeniable impact on the plasma and serum metabolic content. Here, we compared the metabolomic and lipoprotein profiles of blood samples collected at the same time and place from six healthy volunteers but using different collection tubes (each enrolled volunteer provided multiple blood samples at a distance of a few weeks/months): citrate plasma, EDTA plasma, and serum tubes. All samples were analyzed via nuclear magnetic resonance spectroscopy. Several metabolites showed statistically significant alterations among the three blood matrices, and also metabolites' correlations were shown to be affected. The effects of blood collection tubes on the lipoproteins' profiles are relevant too, but less marked. Overcoming the issue associated with different blood collection tubes is pivotal to scale metabolomics and lipoprotein analysis at the level of epidemiological studies based on samples from multicenter cohorts. We propose a statistical solution, based on regression, that is shown to be efficient in reducing the alterations induced by the different collection tubes for both the metabolomic and lipoprotein profiles.

Iron Binding in the Ferroxidase Site of Human Mitochondrial Ferritin.

Ferritins are nanocage proteins that store iron ions in their central cavity as hydrated ferric oxide biominerals. In mammals, further the L (light) and H (heavy) chains constituting cytoplasmic maxi-ferritins, an additional type of ferritin has been identified, the mitochondrial ferritin (MTF). Human MTF (hMTF) is a functional homopolymeric H-like ferritin performing the ferroxidase activity in its ferroxidase site (FS), in which Fe(II) is oxidized to Fe(III) in the presence of dioxygen. To better investigate its ferroxidase properties, here we performed time-lapse X-ray crystallography analysis of hMTF, providing structural evidence of how iron ions interact with hMTF and of their binding to the FS. Transient iron binding sites, populating the pathway along the cage from the iron entry channel to the catalytic center, were also identified. Furthermore, our kinetic data at variable iron loads indicate that the catalytic iron oxidation reaction occurs via a diferric peroxo intermediate followed by the formation of ferric-oxo species, with significant differences with respect to human H-type ferritin.

A framework for validating AI in precision medicine: considerations from the European ITFoC consortium.

BACKGROUND: Artificial intelligence (AI) has the potential to transform our healthcare systems significantly. New AI technologies based on machine learning approaches should play a key role in clinical decision-making in the future. However, their implementation in health care settings remains limited, mostly due to a lack of robust validation procedures. There is a need to develop reliable assessment frameworks for the clinical validation of AI. We present here an approach for assessing AI for predicting treatment response in triple-negative breast cancer (TNBC), using real-world data and molecular -omics data from clinical data warehouses and biobanks. METHODS: The European "ITFoC (Information Technology for the Future Of Cancer)" consortium designed a framework for the clinical validation of AI technologies for predicting treatment response in oncology. RESULTS: This framework is based on seven key steps specifying: (1) the intended use of AI, (2) the target population, (3) the timing of AI evaluation, (4) the datasets used for evaluation, (5) the procedures used for ensuring data safety (including data quality, privacy and security), (6) the metrics used for measuring performance, and (7) the procedures used to ensure that the AI is explainable. This framework forms the basis of a validation platform that we are building for the "ITFoC Challenge". This community-wide competition will make it possible to assess and compare AI algorithms for predicting the response to TNBC treatments with external real-world datasets. CONCLUSIONS: The predictive performance and safety of AI technologies must be assessed in a robust, unbiased and transparent manner before their implementation in healthcare settings. We believe that the consideration of the ITFoC consortium will contribute to the safe transfer and implementation of AI in clinical settings, in the context of precision oncology and personalized care.

Fingerprinting Alzheimer's Disease by 1H Nuclear Magnetic Resonance Spectroscopy of Cerebrospinal Fluid.

In this study, we sought for a cerebrospinal fluid (CSF) metabolomic fingerprint in Alzheimer's disease (AD) patients characterized, according to the clinical picture and CSF AD core biomarkers (Aß42, p-tau, and t-tau), both at pre-dementia (mild cognitive impairment due to AD, MCI-AD) and dementia stages (ADdem) and in a group of patients with a normal CSF biomarker profile (non-AD) using untargeted 1H nuclear magnetic resonance (NMR) spectroscopy-based metabolomics. This is a retrospective study based on two independent cohorts: a Dutch cohort, which comprises 20 ADdem, 20 MCI-AD, and 20 non-AD patients, and an Italian cohort, constituted by 14 ADdem and 12 non-AD patients. 1H NMR CSF spectra were analyzed using OPLS-DA. Metabolomic fingerprinting in the Dutch cohort provides a significant discrimination (86.1% accuracy) between ADdem and non-AD. MCI-AD patients show a good discrimination with respect to ADdem (70.0% accuracy) but only slight differences when compared with non-AD (59.6% accuracy). Acetate, valine, and 3-hydroxyisovalerate result to be altered in ADdem patients. Valine correlates with cognitive decline at follow-up (R = 0.53, P = 0.0011). The discrimination between ADdem and non-AD was confirmed in the Italian cohort. The CSF metabolomic fingerprinting shows a signature characteristic of ADdem patients with respect to MCI-AD and non-AD patients.

Iron Biomineral Growth from the Initial Nucleation Seed in L-Ferritin.

X-ray structures of homopolymeric human L-ferritin and horse spleen ferritin were solved by freezing protein crystals at different time intervals after exposure to a ferric salt and revealed the growth of an octa-nuclear iron cluster on the inner surface of the protein cage with a key role played by some glutamate residues. An atomic resolution view of how the cluster formation develops starting from a (µ3 -oxo)tris[(µ2 -glutamato-κO:κO')](glutamato-κO)(diaquo)triiron(III) seed is provided. The results support the idea that iron biomineralization in ferritin is a process initiating at the level of the protein surface, capable of contributing coordination bonds and electrostatic guidance.

Chemistry at the protein-mineral interface in L-ferritin assists the assembly of a functional (µ3-oxo)Tris[(µ2-peroxo)] triiron(III) cluster.

X-ray structures of homopolymeric L-ferritin obtained by freezing protein crystals at increasing exposure times to a ferrous solution showed the progressive formation of a triiron cluster on the inner cage surface of each subunit. After 60 min exposure, a fully assembled (µ3-oxo)Tris[(µ2-peroxo)(µ2-glutamato-κO:κO')](glutamato-κO)(diaquo)triiron(III) anionic cluster appears in human L-ferritin. Glu60, Glu61, and Glu64 provide the anchoring of the cluster to the protein cage. Glu57 shuttles incoming iron ions toward the cluster. We observed a similar metallocluster in horse spleen L-ferritin, indicating that it represents a common feature of mammalian L-ferritins. The structures suggest a mechanism for iron mineral formation at the protein interface. The functional significance of the observed patch of carboxylate side chains and resulting metallocluster for biomineralization emerges from the lower iron oxidation rate measured in the E60AE61AE64A variant of human L-ferritin, leading to the proposal that the observed metallocluster corresponds to the suggested, but yet unobserved, nucleation site of L-ferritin.

Structural basis of mitochondrial dysfunction in response to cytochrome c phosphorylation at tyrosine 48.

Regulation of mitochondrial activity allows cells to adapt to changing conditions and to control oxidative stress, and its dysfunction can lead to hypoxia-dependent pathologies such as ischemia and cancer. Although cytochrome c phosphorylation-in particular, at tyrosine 48-is a key modulator of mitochondrial signaling, its action and molecular basis remain unknown. Here we mimic phosphorylation of cytochrome c by replacing tyrosine 48 with p-carboxy-methyl-l-phenylalanine (pCMF). The NMR structure of the resulting mutant reveals significant conformational shifts and enhanced dynamics around pCMF that could explain changes observed in its functionality: The phosphomimetic mutation impairs cytochrome c diffusion between respiratory complexes, enhances hemeprotein peroxidase and reactive oxygen species scavenging activities, and hinders caspase-dependent apoptosis. Our findings provide a framework to further investigate the modulation of mitochondrial activity by phosphorylated cytochrome c and to develop novel therapeutic approaches based on its prosurvival effects.

Distal Unfolding of Ferricytochrome c Induced by the F82K Mutation.

It is well known that axial coordination of heme iron in mitochondrial cytochrome c has redox-dependent stability. The Met80 heme iron axial ligand in the ferric form of the protein is relatively labile and can be easily replaced by alternative amino acid side chains under non-native conditions induced by alkaline pH, high temperature, or denaturing agents. Here, we showed a redox-dependent destabilization induced in human cytochrome c by substituting Phe82-conserved amino acid and a key actor in cytochrome c intermolecular interactions-with a Lys residue. Introducing a positive charge at position 82 did not significantly affect the structure of ferrous cytochrome c but caused localized unfolding of the distal site in the ferric state. As revealed by 1H NMR fingerprint, the ferric form of the F82K variant had axial coordination resembling the renowned alkaline species, where the detachment of the native Met80 ligand favored the formation of multiple conformations involving distal Lys residues binding to iron, but with more limited overall structural destabilization.

1H-NMR metabolomics reveals the Glabrescione B exacerbation of glycolytic metabolism beside the cell growth inhibitory effect in glioma.

BACKGROUND: Glioma is the most common and primary brain tumors in adults. Despite the available multimodal therapies, glioma patients appear to have a poor prognosis. The Hedgehog (Hh) signaling is involved in tumorigenesis and emerged as a promising target for brain tumors. Glabrescione B (GlaB) has been recently identified as the first direct inhibitor of Gli1, the downstream effector of the pathway. METHODS: We established the overexpression of Gli1 in murine glioma cells (GL261) and GlaB effect on cell viability. We used 1H-nuclear magnetic resonance (NMR) metabolomic approach to obtain informative metabolic snapshots of GL261 cells acquired at different time points during GlaB treatment. The activation of AMP activated protein Kinase (AMPK) induced by GlaB was established by western blot. After the orthotopic GL261 cells injection in the right striatum of C57BL6 mice and the intranasal (IN) GlaB/mPEG5kDa-Cholane treatment, the tumor growth was evaluated. The High Performance Liquid Chromatography (HPLC) combined with Mass Spectrometry (MS) was used to quantify GlaB in brain extracts of treated mice. RESULTS: We found that GlaB affected the growth of murine glioma cells both in vitro and in vivo animal model. Using an untargeted 1H-NMR metabolomic approach, we found that GlaB stimulated the glycolytic metabolism in glioma, increasing lactate production. The high glycolytic rate could in part support the cytotoxic effects of GlaB, since the simultaneous blockade of lactate efflux with α-cyano-4-hydroxycinnamic acid (ACCA) affected glioma cell growth. According to the metabolomic data, we found that GlaB increased the phosphorylation of AMPK, a cellular energy sensor involved in the anabolic-to-catabolic transition. CONCLUSIONS: Our results indicate that GlaB inhibits glioma cell growth and exacerbates Warburg effect, increasing lactate production. In addition, the simultaneous blockade of Gli1 and lactate efflux amplifies the anti-tumor effect in vivo, providing new potential therapeutic strategy for this brain tumor.

High-Throughput Metabolomics by 1D NMR.

Metabolomics deals with the whole ensemble of metabolites (the metabolome). As one of the -omic sciences, it relates to biology, physiology, pathology and medicine; but metabolites are chemical entities, small organic molecules or inorganic ions. Therefore, their proper identification and quantitation in complex biological matrices requires a solid chemical ground. With respect to for example, DNA, metabolites are much more prone to oxidation or enzymatic degradation: we can reconstruct large parts of a mammoth's genome from a small specimen, but we are unable to do the same with its metabolome, which was probably largely degraded a few hours after the animal's death. Thus, we need standard operating procedures, good chemical skills in sample preparation for storage and subsequent analysis, accurate analytical procedures, a broad knowledge of chemometrics and advanced statistical tools, and a good knowledge of at least one of the two metabolomic techniques, MS or NMR. All these skills are traditionally cultivated by chemists. Here we focus on metabolomics from the chemical standpoint and restrict ourselves to NMR. From the analytical point of view, NMR has pros and cons but does provide a peculiar holistic perspective that may speak for its future adoption as a population-wide health screening technique.

Electrostatic and Structural Bases of Fe2+ Translocation through Ferritin Channels.

Ferritin molecular cages are marvelous 24-mer supramolecular architectures that enable massive iron storage (>2000 iron atoms) within their inner cavity. This cavity is connected to the outer environment by two channels at C3 and C4 symmetry axes of the assembly. Ferritins can also be exploited as carriers for in vivo imaging and therapeutic applications, owing to their capability to effectively protect synthetic non-endogenous agents within the cage cavity and deliver them to targeted tissue cells without stimulating adverse immune responses. Recently, X-ray crystal structures of Fe2+-loaded ferritins provided important information on the pathways followed by iron ions toward the ferritin cavity and the catalytic centers within the protein. However, the specific mechanisms enabling Fe2+ uptake through wild-type and mutant ferritin channels is largely unknown. To shed light on this question, we report extensive molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements that characterize the transport properties and translocation mechanism of Fe2+ through the two ferritin channels, using the wild-type bullfrog Rana catesbeiana H' protein and some of its variants as case studies. We describe the structural features that determine Fe2+ translocation with atomistic detail, and we propose a putative mechanism for Fe2+ transport through the channel at the C3 symmetry axis, which is the only iron-permeable channel in vertebrate ferritins. Our findings have important implications for understanding how ion permeation occurs, and further how it may be controlled via purposely engineered channels for novel biomedical applications based on ferritin.

Unsaturated Long-Chain Fatty Acids Are Preferred Ferritin Ligands That Enhance Iron Biomineralization.

Ferritin is a ubiquitous nanocage protein, which can accommodate up to thousands of iron atoms inside its cavity. Aside from its iron storage function, a new role as a fatty acid binder has been proposed for this protein. The interaction of apo horse spleen ferritin (HoSF) with a variety of lipids has been here investigated through NMR spectroscopic ligand-based experiments, to provide new insights into the mechanism of ferritin-lipid interactions, and the link with iron mineralization. 1D 1 H, diffusion (DOSY) and saturation-transfer difference (STD) NMR experiments provided evidence for a stronger interaction of ferritin with unsaturated fatty acids compared to saturated fatty acids, detergents, and bile acids. Mineralization assays showed that oleate c aused the most efficient increase in the initial rate of iron oxidation, and the highest formation of ferric species in HoSF. The comprehension of the factors inducing a faster biomineralization is an issue of the utmost importance, given the association of ferritin levels with metabolic syndromes, such as insulin resistance and diabetes, characterized by fatty acid concentration dysregulation. The human ferritin H-chain homopolymer (HuHF), featuring ferroxidase activity, was also tested for its fatty acid binding capabilities. Assays show that oleate can bind with high affinity to HuHF, without altering the reaction rates at the ferroxidase site.

Investigation of the Iron(II) Release Mechanism of Human H-Ferritin as a Function of pH.

We investigated the kinetics of the release of iron(II) ions from the internal cavity of human H-ferritin as a function of pH. Extensive molecular dynamics simulations of the entire 24-mer ferritin provided atomic-level information on the release mechanism. Double protonation of His residues at pH 4 facilitates the removal of the iron ligands within the C3 channel through the formation of salt bridges, resulting in a significantly lower release energy barrier than pH 9.

Individual Human Metabolic Phenotype Analyzed by (1)H NMR of Saliva Samples.

Saliva is an important physiological fluid that contains a complex mixture of analytes that may produce a characteristic individual signature. In recent years, it has been demonstrated that urine possesses a clear signature of the individual metabolic phenotype. Here NMR-based metabolomics was employed to analyze saliva from 23 healthy volunteers. About six saliva samples were collected daily from each individual for 10 consecutive days: 7 days in a real-life situation (days 1-7, Phase I) and 3 days (days 8-10, Phase II) under a standardized diet plus a physical exercise program at day 10. The result is the first demonstration of the existence of an individual metabolic phenotype in saliva. A systematic comparative analysis with urine samples from the same collection scheme demonstrates that the individual phenotype in saliva is slightly weaker than that in urine but less influenced by diet.

Is His54 a gating residue for the ferritin ferroxidase site?

Ferritin is a ubiquitous iron concentrating nanocage protein that functions through the enzymatic oxidation of ferrous iron and the reversible synthesis of a caged ferric-oxo biomineral. Among vertebrate ferritins, the bullfrog M homopolymer ferritin is a frequent model for analyzing the role of specific amino acids in the enzymatic reaction and translocation of iron species within the protein cage. X-ray crystal structures of ferritin in the presence of metal ions have revealed His54 binding to iron(II) and other divalent cations, with its imidazole ring proposed as "gate" that influences iron movement to/from the active site. To investigate its role, His54 was mutated to Ala. The H54A ferritin variant was expressed and its reactivity studied via UV-vis stopped-flow kinetics. The H54A variant exhibited a 20% increase in the initial reaction rate of formation of ferric products with 2 or 4 Fe²âº/subunit and higher than 200% with 20 Fe²âº/subunit. The possible meaning of the increased efficiency of the ferritin reaction induced by this mutation is proposed taking advantage of the comparative sequence analysis of other ferritins. The data here reported are consistent with a role for His54 as a metal ion trap that maintains the correct levels of access of iron to the active site. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.