Members of the nucleobase ascorbate transporter (NAT) family (SLC23) are elevator-type transporters that are responsible for the uptake of nucleobases and ascorbate. In fungi, NAT members are also responsible for the specific uptake of antifungal nucleobase analogues, such as oxypurinol, allopurinol, or 8-azaguanine. Here, we report nearly full-length cryo-EM structures of UapA, a high-affinity purine transporter from the model fungus Aspergillus nidulans, in inward-facing apo- and substrate-loaded conformations at 2.06 to 3.5 Å in detergent and lipid nanodiscs. The high-resolution structures reveal the role of water molecules and lipids in substrate binding, specificity, transporter dimerization, and activity. Notably, the N-tail of UapA is found to be structured, interacting with both the core and scaffold domains, which in combination with functional data suggests a dual role in trafficking and transport dynamics. Overall, our study provides unprecedented structural and functional insights into an elevator-type fungal transporter, which may well contribute to the exploitation of NAT transporters as specific gateways for targeted pharmacological antifungal approaches.

Regardless of the massive global efforts to combat the virus causing SARS-CoV-2 syndrome, the infection remains a substantial health challenge worldwide in the years following 2019. To this direction, targeting the main viral protease Mpro has been proposed as a tractable and particularly promising approach toward developing effective and safe COVID-19 antivirals. By applying an integrated workflow combining a previously developed in silico consensus ranking protocol with two orthogonal in vitro methods, the NCI/DTP repository is screened and the discovery of an original naphthol scaffold with Mpro inhibitory properties is reported. The hit is characterized in terms of structure and binding thermodynamics by combining X-ray crystallography and isothermal titration calorimetry where a binding affinity constant of 1.55 μM is determined. The compound is further evaluated against virus-infected cells, where an EC50 value of 7.23 μM and comparable toxicity with nirmatrelvir are measured. Chemical synthesis is additionally employed to facilitate optimal exploration of the structure-activity relationship landscape regarding the new hit. By integrating computational, biophysical, and enzymatic methods, the suggested approach allows the combination of a structural hypothesis with functional evidence and shows its capacity toward identifying and rationally optimizing structurally original noncovalent Mpro inhibitors.

Numerous genetic variants have been identified by genome-wide association studies as being associated with colorectal cancer (CRC) risk. Metabolome-wide association analysis was performed for 187 CRC-associated genetic variants using genomic data and untargeted 1H nuclear magnetic resonance urine metabolomics from 1951 Airwave Health Monitoring Study participants. We identified statistically significant associations between seven CRC single-nucleotide polymorphisms (SNPs) and urinary metabolites. This included SNPs within or close to RHPN2 with sucrose (P = 1.2 × 10-7), SLC6A18 with amino acids (P = 6.9 × 10-5 with tyrosine, P = 9.9 × 10-5 with leucine), and MAP2K5 and BMP2 with gut microbial metabolites (P = 1.6 × 10-4 and P = 4.4 × 10-4). The most significant correlation was followed by functional experiments in Caco-2 colon cancer cells. CRISPR-mediated knockout of a 48-nt RHPN2 intronic region containing rs10411210 in colon cancer cells compromised cell growth. RNA sequencing was performed in the two sets of clones (3 edited and 3 unedited) followed by pathway enrichment, and gene ontology analysis depicted extensive deregulation of genes (448 up- and 195 downregulated) involved in cell division and several metabolic processes. Overall, these findings demonstrate that integrating genetic and metabolomic data highlights the importance of the RHPN2 intronic locus in CRC potentially through metabolic processes affecting excretion of dietary and other metabolites.

Despite the renewed interest in streptothricin as a potential therapeutic, only a few streptothricin acetyltransferases have been characterized to date. In the present study, genome analysis of Streptomyces rochei ATHUBA 263 revealed two putative streptothricin acetyltransferase genes, srstat1 and srstat2. The genes were heterologously expressed in Escherichia coli, and the enzymes were purified for biochemical characterization. Both SrStat1 and SrStat2 conferred high-level resistance to streptothricin in E. coli and catalyzed streptothricin acetylation in vitro efficiently, with SrStat1 showing slightly higher substrate affinity and catalytic efficiency than SrStat2. Computational structural analysis combining AlphaFold-based modeling and molecular dynamics simulations demonstrated the conserved active site residues and revealed some subtle differences in active site architecture of the two enzymes. In addition, both enzymes displayed a weaker acetylation activity toward chloramphenicol in vitro. NMR spectroscopy provided evidence that acetylation occurs at the hydroxyl group located at the C-3 position of chloramphenicol, demonstrating that these enzymes are capable of O-acetylation in addition to the N-acetylation. Together, these findings expand the set of characterized streptothricin acetyltransferases and highlight the broad substrate specificity of GNATs. These insights provide valuable context for resistance mechanisms associated with streptothricin and may contribute to the renewed interest in streptothricin F as a therapeutic option against multidrug-resistant pathogens.

The tandem Tudor domain (TTD) of UHRF1 is a compelling epigenetic target for novel cancer therapeutics. Here, we integrate theoretical simulations, sophisticated biophysical evaluations and cellular assays to characterize potent TTD binders. Screening of small drug- and fragment-like collections identifies several TTD ligands, with the most promising hit being the local anesthetic hydroxyprocaine. The ligand is characterized in terms of its binding requisites by calorimetry, affording a Kd of 1.46 μM and a well-balanced thermodynamic profile. Molecular dynamics simulations combined with heat capacity measurements and osmotic stress titrations confirm that hydroxyprocaine binds stably to the TTD by displacing approximately 26 interfacial water molecules upon complexation. A targeted follow-up screen focusing on sodium channel blockers yields two additional, although less promising hits, mexiletine and triamterene. In the DU145 prostate cancer cell line, hydroxyprocaine treatment significantly up-regulates key downstream targets including the tumor-suppressor p53 and, to a lesser degree, the stress and inflammation regulators p38 and p65, respectively, while exhibiting very low cytotoxicity. Finally, a previously undocumented interdomain interaction between TTD and its N-terminal adjacent Ubiquitin-like domain is reported, introducing a novel, potentially druggable UHRF1 regulatory feature. Together, these findings establish hydroxyprocaine as a highly viable chemical scaffold for TTD-targeted drug development.

Natural products represent an important source of bioactive compounds; however, their complexity often challenges conventional discovery approaches. In this study, an ethyl acetate extract of Artemisia arborescens (Vaill.) L. was investigated prior to any isolation using an integrated statistical spectroscopy-driven dereplication strategy combining the HeteroCovariance Approach (HetCA), Statistical Total Correlation Spectroscopy (STOCSY), 1H NMR, and LC-HRMS/MS. HetCA illustrated candidate features correlated with antileishmanial activity, predominantly associated with furofuran lignans. STOCSY facilitated metabolite annotation and distinction of structurally similar metabolites, enabling the identification of a novel lignan and a previously unreported compound in A. arborescens. Targeted isolation verified STOCSY-guided annotations, while biological evaluation of the pure metabolites revealed that compounds 1-3 exhibit moderate activity against Leishmania promastigotes, despite originating from a crude extract exhibiting negligible antileishmanial activity under the initial screening conditions. These findings demonstrate that HetCA can reveal bioactive compounds independently of extract-level activity and highlight its potential as a robust tool for broadening natural product drug discovery beyond conventional bioactivity-guided approaches.

3-mercaptopyruvate sulfurtransferase (MPST) is an enzyme implicated in the generation of the gasotransmitter hydrogen sulfide (H2S). Unlike, the other two H2S-synthesizing enzymes cystathionine gamma lyase (CSE) and cystathionine beta synthase (CBS), MPST is found in the mitochondria. However, the mechanisms through which MPST gains access to the mitochondria and its exact localization within this organelle remain unclear. Using immunogold electron microscopy staining, we localized MPST on the inner mitochondrial membrane. To study the pathway of mitochondrial entry for MPST, pharmacological inhibitors of different components of the translocase of outer/inner membrane were used. In line with the observation that ΜPST is found on the inner mitochondrial membrane, inhibition of TIM23 blocked MPST mitochondrial entry. Generation of N-terminally truncated forms of ΜPST did not interfere with the ability of the enzyme to gain access into the mitochondria, suggesting that an N-terminal pre-sequence does not mediate MPST mitochondrial entry. In agreement to this finding, cytosolic and mitochondrial MPST had a similar molecular weight. Interestingly, N-terminally deleted MPST exhibited reduced expression levels, indicating that this part of the enzyme is required for protein stability. Molecular dynamics simulations confirmed that deletion of the first 39 amino acids of the enzyme destabilizes the protein. Our findings reveal that MPST is present on the inner mitochondrial membrane and that its entry into mitochondria does not involve the N-terminus of the protein.

Background/Objectives: The rapid evolution of bacterial resistance and the high cost of drug development have attributed greatly to the dearth in drug design. Computational approaches and natural product exploitation offer potential solutions to accelerate drug discovery. Methods: In this research article, we aimed to identify novel antibacterial hits. For the in silico studies, molecular scaffolds from the in-house chemical library of the Department of Pharmacy of Athens (Pharmalab) and the National Cancer Institute (NCI) were screened and selected for further experimental procedures. Compounds from both libraries that were not previously screened for their antimicrobial properties were tested in vitro against Gram-positive and Gram-negative bacterial strains. The microdilution method was used to determine the minimum inhibitory concentrations (MICs). Results: In silico screening identified twenty promising molecules from the NCI and seven from the Pharmalab databases. The unexplored compounds for their antibacterial activity can be characterized as weak strain-specific antimicrobials. The NSC 610491 and NSC 610493 were active against Staphylococcus aureus (MIC: 25 and 12.5 µg/mL, respectively) and methicillin-resistant S. aureus (MRSA) (MIC: 50 and 12.5 µg/mL, respectively). Six out of seven hydroxytyrosol (HTy) compounds were moderately active (MIC: 25–50 µg/mL) against S. aureus, MRSA and Enterococcus faecalis. For the Gram-negative bacteria, no activity was detected (≥100 µg/mL). Conclusions: The tested scaffolds could be considered as promising candidates for novel antimicrobials with improvements. Further experimentation is required to assess mechanisms of action and evaluate the efficacy and safety.

Purpose:Sirtuins (SIRTs) play a critical role in redox and metabolic regulation of the myocardium; however, the cardioprotective potential of SIRT5 in terms of infarct size (IS) reduction is still elusive. Herein, we employed the newly synthesized SIRT5-specific agonist, MC3215, developed by our group, to explore for the first time the pharmacological activation of SIRT5 as a target for cardioprotection. Methods and Results:In in vitro screening experiments, SIRT1 and SIRT5 agonists, namely, MC2606 and MC3215, at 1-20 μΜ were added to cardiomyoblasts (H9c2) and human endothelial cells (EA.hy-926) during 24 h hypoxia/2 h reoxygenation (H/R). SIRT1 and SIRT5 agonists mitigated H/R injury. Male C57BL/6J mice underwent 30 min ischemia (I) followed by 2 h or 24 h reperfusion (R). Mice received vehicle, the SIRT1 or SIRT5 agonists at 20 and 30 mg/kg at the 20th min of ischemia, and IS was quantified via triphenyl-tetrazolium chloride staining (n=5-7/group). MC3215-mediated SIRT5 activation reduced IS at 24 h R at 20mg/kg compared to controls (25.18±2.7% vs 38.80±4.7%). MC3215 treatment resulted in reduced protein malonylation in all experimental settings. Targeted mass-spectrometry-based metabolomics in the ischemic heart at the 10th min of R suggested increased fatty acid oxidation, as indicated by increased N3-Trimethyllysine and D-pantothenate. Concomitantly, molecular analysis indicated that the SIRT5 agonist activated AMPKα and Reperfusion Injury Salvage Kinase (RISK) pathway. Additionally, at 3 h reperfusion, MC3215 led to increased mitofusin 2 without altering apoptosis, paving towards improved mitochondrial dynamics. Co-administration of SIRT5 inhibitor, TW-37, abrogated MC3215-mediated cardioprotection. Conclusion:SIRT5 pharmacological agonism emerges as a novel cardioprotective target, leading to RISK pathway activation and mitochondria-related metabolic effects, converging at salvaging ischemic myocardium from I/R injury.

Natural product drug discovery faces important challenges related to efficient complex mixture analysis. To address this, dereplication strategies have been developed to enhance and accelerate the detection of bioactive compounds in plant extracts. Among these, statistical spectroscopy-based methods HeteroCovariance Approach (HetCA) and Statistical Total Correlation Spectroscopy (STOCSY) have been proposed. In this study, HetCA and STOCSY are applied to Artemisia umbelliformis subsp. eriantha extract, to discover antileishmanial compounds. Spectral data from appropriately generated extract fractions were statistically correlated with antileishmanial activity results. HetCA along with STOCSY revealed sets of NMR signals correlated to the activity and led to the identification of specific bioactive compounds. These components were purposefully isolated and tested against L. infantum promastigotes, designating that the positively correlated compounds 5-deoxy-5-hydroperoxytelekin 1 and telekin 2 exert strong antileishmanial effects, with a half-maximal effective concentration (EC50) of 4.5 and 5.1 μΜ, respectively. Their toxicity was also tested on model host cells, THP-1 macrophages, yielding a 50 % cytotoxic concentration of 29.7 μΜ and 15 μΜ, respectively. For verification purposes, non-correlated compounds were also isolated and didn't show significant antileishmanial activity. Overall, the combination of HetCA and STOCSY was able to identify the most active compounds, facilitating the detection of bioactive components and opening new insights into natural product drug discovery.

The Set and Ring domain of the UHRF1 oncogene is responsible for its interaction with hemimethylated DNA and faithful propagation of epigenetic signaling over cellular replication. Inhibiting this recognition can have serious implications for UHRF1 functionality and may possibly enable therapeutic interventions. Based on a previous finding indicating a promising in vitro DNA demethylating potential of a pyrimidine derivative, a subscaffold search was performed in the NCI/DTP compound repository to discover similar molecules and evaluate their affinity for the SRA domain of UHRF1. Toward this direction, several compounds were evaluated using a thermal melt screen, and the most promising hits were subsequently studied by calorimetry in terms of their capacity to bind the 5-methylcytosine recognition site of UHRF1. A markedly different thermodynamic profile between the two confirmed hits with an intense enthalpy-entropy compensation signature was determined. The systems were further studied by biased and unbiased molecular simulations, computational hydration mapping, and calorimetry-based heat capacity measurements to devise a hypothesis on the structural requisites for efficient SRA binding. The most potent compound was evaluated for its DNA methylation effects against the UHRF1-dependent colorectal cancer HCT116 cells, where promising global demethylating activity reaching an approximate 75% reduction compared to control was achieved after treatment with 25 μM of NSC232005. Based on the presented results, rationally substituted analogues of the uracil scaffold appear as highly promising UHRF1 modulators for exploring its diverse functionalities and validating the protein as a drug target.

IntroductionRheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and pain. This study investigates plasma lipoprotein and glycoprotein profiles in RA patients to identify clinically relevant markers for disease monitoring.MethodsLipoprotein composition and subfractions were analyzed in plasma from 161 RA patients and 46 controls using proton nuclear magnetic resonance (1H NMR) spectroscopy (Lipoprotein Subclass Analysis (B.I.LISA) platform) along with N-acetylglycoprotein signals GlycA and GlycB. Lipoprotein subclasses and glycoproteins in RA and disease-modifying anti-rheumatic drug (DMARD)-naive RA patients were compared to controls, and comprehensive profiles were evaluated in activity and remission. Correlations with disease activity score (DAS28), inflammation marker C-reactive protein (CRP), and Visual Analogue Scale (VAS) of pain were assessed using regression models, adjusting for age, gender, and CVD.ResultsRA patients exhibited a distinct lipoprotein and glycoprotein profile, with increased triglycerides, cholesterol, apolipoproteins (A1, A2, B100), and changes in LDL, HDL, GlycA, and GlycB. Glycoproteins were significantly higher in DMARD-naive RA (AUC ≈ 0.9) validating these NMR signals as biomarkers of inflammation. Patients in remission had higher small dense HDL and lower LDL-triglycerides than those with high disease activity. VAS correlated with LDL-triglycerides, while DAS28 correlated with small dense LDL-triglycerides and glycoproteins, inversely with large LDL, small HDL lipids. H4A1 alone characterizes RA remission (AUC ≈ 0.8).ConclusionLipoprotein profiles in RA correlate with disease activity, inflammation, and pain. Large HDL, intermediate LDL and glycoproteins serve for RA monitoring as well as potential molecular markers of pain.

FurE is a H+ symporter specific for the cellular uptake of uric acid, allantoin, uracil, and toxic nucleobase analogues in the fungus Aspergillus nidulans. Being member of the NCS1 protein family, FurE is structurally relat-ed to the APC-superfamily of transporters. APC-type transporters are charac-terised by a 5+5 inverted repeat fold made of ten transmembrane segments (TMS1-10) and function through the rocking-bundle mechanism. Most APC-type transporters possess two extra C-terminal TMS segments (TMS11-12), the function of which remains elusive. Here we present a systematic muta-tional analysis of TMS11-12 of FurE and show that two specific aromatic resi-dues in TMS12, Trp473 and Tyr484, are essential for ER-exit and trafficking to the plasma membrane (PM). Molecular modeling shows that Trp473 and Tyr484 might be essential through dynamic interactions with residues in TMS2 (Leu91), TMS3 (Phe111), TMS10 (Val404, Asp406) and other aromatic residues in TMS12. Genetic analysis confirms the essential role of Phe111, Asp406 and TMS12 aromatic residues in FurE ER-exit. We further show that co-expression of FurE-Y484F or FurE-W473A with wild-type FurE leads to a dominant negative phenotype, compatible with the concept that FurE mole-cules oligomerize or partition in specific microdomains to achieve concentra-tive ER-exit and traffic to the PM. Importantly, truncated FurE versions lacking TMS11-12 are unable to reproduce a negative effect on the trafficking of co-expressed wild-type FurE. Overall, we show that TMS11-12 acts as an intra-molecular chaperone for proper FurE folding, which seems to provide a struc-tural code for FurE partitioning in ER-exit sites.

Background: Cu/Zn Superoxide Dismutase 1 (SOD1) is a 32 kDa cytosolic dimeric metalloenzyme that neutralizes superoxide anions into oxygen and hydrogen peroxide. Mutations in SOD1 are associated with ALS, a disease causing motor neuron atrophy and subsequent mortality. These mutations exert their harmful effects through a gain of function mechanism, rather than a loss of function. Despite extensive research, the mechanism causing selective motor neuron death still remains unclear. A defining feature of ALS pathogenesis is protein misfolding and aggregation, evidenced by ubiquitinated protein inclusions containing SOD1 in affected motor neurons. This work aims to identify compounds countering SOD1(A4V) misfolding and aggregation, which could potentially aid in ALS treatment. Methods: The approach employed was in vitro screening of a library comprising 1280 pharmacologically active compounds (LOPAC®) in the context of drug repurposing. Using differential scanning fluorimetry (DSF), these compounds were tested for their impact on SOD1(A4V) thermal stability. Results and Conclusions: Dimer stability was the parameter chosen as the criterion for screening, since the dissociation of the native SOD1 dimer is the step prior to its in vitro aggregation. The screening revealed one compound raising protein-ligand Tm by 6 °C, eleven inducing a higher second Tm, suggesting a stabilization effect, and fourteen reducing Tm from 10 up to 26 °C, suggesting possible interactions or non-specific binding.

Background: Sjögren’s disease (SjD) is a systemic autoimmune disorder that primarily affects the exocrine glands, particularly the salivary and lacrimal glands. Recent efforts have exploited serum and saliva metabolome analysis to enhance our understanding of pathogenetic mechanisms and the development of novel biomarkers, however these studies are hampered by limited patient numbers, poorly characterized SjD cases, the absence of proper control groups, a lack of clinicohistologic associations, and variations in the metabolomic analysis techniques used. Objectives: The aim of the current study is to enlighten the latent biochemical background of SjD and to identify potential biomarkers facilitating early non-interventional SjD diagnosis. Methods: To investigate metabolomic profiles associated with SjD, we conducted a comprehensive analysis using saliva samples from patients presenting with sicca symptoms. All patients diagnosed with SjD fulfil the 2016 ACR-EULAR classification criteria and all minor salivary gland biopsies were re-evaluated by the same expert on SjD salivary gland pathology. Patients not fulfilling the criteria for SjD were classified as sicca controls (n=21 for QTOF and 17 for 1H NMR). Our metabolomics profiling protocol allows for a simultaneous, non-targeted measurement of a wide variety of small molecules, exploiting 2 different techniques: high-resolution mass spectrometry (QTOF) and high-resolution 1H 1D NMR spectroscopy. Patients were categorized based on their focus score (FS) into low (LFS, FS<1, n= 8 f for QTOF and 6 for 1H NMR), medium (MFS, 13, n= 9 for QTOF and 9 for 1H NMR) groups. In our analysis, we considered potential influences of gender, age, saliva flow rate during sample collection, and FS. Multi- and univariate statistical methodologies were applied for group classification and biomarker detection. Partial Least Squares Projection to Latent Structures modeling (PLS; SIMCA v. 14, MKS Umetrics AB) was used to correlate spectroscopic data with Focus score values. MetaboAnalyst, a web-based platform for metabolomic data analysis and interpretation was also used. Results: Utilizing multivariate statistical methodologies, patients were successfully categorized into Low, Moderate, and High Focus Score (LFS, MFS, HFS) groups, as well as into Sjögren and Non-Sjögren diagnostic categories, demonstrating acceptable model performance. Preliminary analyses identified upregulated metabolites in Sjögren patients saliva, including Choline (Fold Change, FC=1.3), Lactic acid (FC=1.4), Taurine (FC=1.1) from the 1H NMR analysis, and Neuraminic acid (FC=2.2), Deoxyinosine (FC=2.8), Pimelylcarnitine (FC=1.8) from the QTOF analysis. In HFS patients, elevated levels of Leucine (FC=1.3), Indoleacrylic acid (FC=1.2), Guanidinoacetic acid (FC=1.3) were observed, while Diaminopropionic acid (FC=1.4) and Prostaglandin E3 (FC=1.3) were decreased. Proof of concept correlation between the metabolic fingerprint and focus score values revealed a high correlation (R2=0.94) with strong statistical significance (p=9.7 × 10−6). Conclusion: To the best of our knowledge, this is the first study investigating saliva sample metabolomics with 2 different techniques, coupled with biopsy FS data. This approach provides an insight into the distinctive alterations characterizing both sicca controls and Sjögren’s disease patients across different disease stages. According to the findings the intensity of inflammation, as depicted by the salivary biopsy focus score, is highly associated with the salivary metabolome landscape. Meanwhile some metabolites presen in the saliva have emerged as novel biomarkers aiming to offer a non-invasive tool to support SjD clinical diagnosis and histologic classification. Further studies are needed to validate these results and establish the clinical utility in SjD. REFERENCES: NIL. Acknowledgements: NIL. Disclosure of Interests: None declared.Figure 1

Foodomics employs advanced analytical techniques to provide answers regarding food composition, authenticity control, marker identification and issues related to food quality and safety. Nuclear magnetic resonance (NMR) spectroscopy and chromatography hyphenated to mass spectrometry (MS) are the main analytical platforms used in this field. Nevertheless, they are rarely employed in an integrated manner, and even then, the contribution of each technique remains vague. Table olives (Olea europaea L.) are a food commodity of high economic and nutritional value with an increasing production tendency over the last two decades, which, however, suffers from extensive fraud incidents and quality determination uncertainties. Thus, the current attempt aims towards two axes with the first being the multilevel integration of LC-HRMS and NMR data of the same samples and table olives being the selected matrix. In more detail, UPLC-HRMS/MS-based analysis was compared at different stages within an untargeted metabolomics workflow with an NMR-based study and the complementarity of the two platforms was evaluated. Furthermore, statistical heterospectroscopy (SHY), rarely employed in foodomics, combining the spectroscopic with spectrometric datasets and aiming to increase the confidence level of annotated biomarkers was applied. Amongst these lines, the second parallel axis of this study was the detailed characterization of table olives' metabolome in search for quality markers considering the impact of geographical (from Northern to Southern Greece) and botanical origin (Kalamon, Konservolia, Chalkidikis cultivars), as well as processing parameters (Spanish, Greek). To that end, using deep dereplication tools including statistical methods, with SHY employed for the first time in table olives, different biomarkers, belonging to the classes of phenyl alcohols, phenylpropanoids, flavonoids, secoiridoids and triterpenoids were identified as responsible for the observed classifications. The current binary pipeline, focusing on biomarkers’ identification confidence, could be suggested as a meaningful workflow not only in olive-based products, but also in food quality control and foodomics in general.

Abstract: Giant Cell Arteritis (GCA) is an autoimmune/autoinflammatory disease affecting large vessels in patients over 50 years old. The disease presents as an acute inflammatory response with two phenotypes, cranial-GCA and Large Vessel Vasculitis (LV)-GCA, involving the thoracic aor-ta and its branches. 18F-fluorodeoxyglucose positron emission tomography-computed tomogra-phy (18F-FDG PET-CT) is among the imaging techniques contributing to diagnosis of patients with systemic disease. However, its association with soluble inflammatory markers is still elu-sive. This proof-of-concept study aims to identify novel soluble serum biomarkers in PET/CT pos-itive patients with LV-GCA and associate them with active (0 months) and inactive disease (6 months following treatment), in sequential samples. The Most-Disease-Segment Target-to-Background Ratio (TBRMDS) was calculated for 9 LV-GCA patients, while 12 cranial-GCA and 7 Polymyalgia Rheumatica patients with negative initial PET/CT served as disease controls. Serum macrophage-related cytokines were evaluated by Cytometric Bead Array (CBA). Finally, previously published NMR-metabolomics data acquired at the same blood sampling were associated with PET/CT findings. TBRMDS was significantly increased in active versus inactive disease (3.45 vs. 2.55, p=0.008). The analysis identified 6 serum metabolites, as more sensitive to change from the active to inactive state. Among them, choline levels were exclusively altered in the LV-GCA group, but not the disease controls. Cytokine levels were not associated with PET/CT activity. Combining with CRP, ESR, and TBRMDS a composite index was generated to depict well the differences between active and inactive systemic LV-GCA (25.45 vs 11.45, p=0.0039). These preliminary results could pave the way for more extensive studies integrating serum metabolomic parameters with PET/CT imaging data to extract sensitive composite disease indexes useful for the everyday clinical practice.

Giant cell arteritis (GCA) is an autoimmune/autoinflammatory disease affecting large vessels in patients over 50 years old. The disease presents as an acute inflammatory response with two phenotypes, cranial GCA and large-vessel vasculitis (LV)-GCA, involving the thoracic aorta and its branches. 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET-CT) is among the imaging techniques contributing to diagnosing patients with systemic disease. However, its association with soluble inflammatory markers is still elusive. This proof-of-concept study aims to identify novel soluble serum biomarkers in PET/CT-positive patients with LV-GCA and associate them with active (0 months) and inactive disease (6 months following treatment), in sequential samples. The most-diseased-segment target-to-background ratio (TBRMDS) was calculated for 13 LV-GCA patients, while 14 cranial GCA and 14 Polymyalgia Rheumatica patients with negative initial PET/CT scans served as disease controls. Serum macrophage-related cytokines were evaluated by cytometric bead array (CBA). Finally, previously published NMR/metabolomics data acquired from the same blood sampling were analyzed along with PET/CT findings. TBRMDS was significantly increased in active versus inactive disease (3.32 vs. 2.65, p = 0.006). The analysis identified nine serum metabolites as more sensitive to change from the active to inactive state. Among them, choline levels were exclusively altered in the LV-GCA group but not in the disease controls. Cytokine levels were not associated with PET/CT activity. Combining CRP, ESR, and TBRMDS with choline levels, a composite index was generated to distinguish active and inactive LV-GCA (20.4 vs. 11.62, p = 0.001). These preliminary results could pave the way for more extensive studies integrating serum metabolomic parameters with PET/CT imaging data to extract sensitive composite disease indexes useful for everyday clinical practice.