Stress-Induced Metabolic Disorder in Peripheral CD4+ T Cells Leads to Anxiety-like Behavior.

Physical or mental stress leads to neuroplasticity in the brain and increases the risk of depression and anxiety. Stress exposure causes the dysfunction of peripheral T lymphocytes. However, the pathological role and underlying regulatory mechanism of peripheral T lymphocytes in mood disorders have not been well established. Here, we show that the lack of CD4+ T cells protects mice from stress-induced anxiety-like behavior. Physical stress-induced leukotriene B4 triggers severe mitochondrial fission in CD4+ T cells, which further leads to a variety of behavioral abnormalities including anxiety, depression, and social disorders. Metabolomic profiles and single-cell transcriptome reveal that CD4+ T cell-derived xanthine acts on oligodendrocytes in the left amygdala via adenosine receptor A1. Mitochondrial fission promotes the de novo synthesis of purine via interferon regulatory factor 1 accumulation in CD4+ T cells. Our study implicates a critical link between a purine metabolic disorder in CD4+ T cells and stress-driven anxiety-like behavior.

8-oxoguanine riboswitches in bacteria detect and respond to oxidative DNA damage.

Riboswitches rely on structured aptamer domains to selectively sense their target ligands and regulate gene expression. However, some riboswitch aptamers in bacteria carry mutations in their otherwise strictly conserved binding pockets that change ligand specificities. The aptamer domain of a riboswitch class originally found to selectively sense guanine forms a three-stem junction that has since been observed to exploit numerous alterations in its ligand-binding pocket. These rare variants have modified their ligand specificities to sense other purines or purine derivatives, including adenine, 2'-deoxyguanosine (three classes), and xanthine. Herein, we report the characteristics of a rare variant that is narrowly distributed in the Paenibacillaceae family of bacteria. Known representatives are always associated with genes encoding 8-oxoguanine deaminase. As predicted from this gene association, these variant riboswitches tightly bind 8-oxoguanine (8-oxoG), strongly discriminate against other purine derivatives, and function as genetic "ON" switches. Following exposure of cells to certain oxidative stresses, a representative 8-oxoG riboswitch activates gene expression, likely caused by the accumulation of 8-oxoG due to oxidative damage to G nucleobases in DNA, RNA, and the nucleotide pool. Furthermore, an engineered version of the variant aptamer was prepared that exhibits specificity for 8-oxoadenine, further demonstrating that RNA aptamers can acquire mutations that expand their ability to detect and respond to oxidative damage.

Variants of the guanine riboswitch class exhibit altered ligand specificities for xanthine, guanine, or 2'-deoxyguanosine.

The aptamer portions of previously reported riboswitch classes that sense guanine, adenine, or 2'-deoxyguanosine are formed by a highly similar three-stem junction with distinct nucleotide sequences in the regions joining the stems. The nucleotides in these joining regions form the major features of the selective ligand-binding pocket for each aptamer. Previously, we reported the existence of additional, rare variants of the predominant guanine-sensing riboswitch class that carry nucleotide differences in the ligand-binding pocket, suggesting that these RNAs have further diversified their structures and functions. Herein, we report the discovery and analysis of three naturally occurring variants of guanine riboswitches that are narrowly distributed across Firmicutes. These RNAs were identified using comparative sequence analysis methods, which also revealed that some of the gene associations for these variants are atypical for guanine riboswitches or their previously known natural variants. Binding assays demonstrate that the newfound variant riboswitch representatives recognize xanthine, guanine, or 2'-deoxyguanosine, with the guanine class exhibiting greater discrimination against related purines than the more common guanine riboswitch class reported previously. These three additional variant classes, together with the four previously discovered riboswitch classes that employ the same three-stem junction architecture, reveal how a simple structural framework can be diversified to expand the range of purine-based ligands sensed by RNA.

Single substrate-functionalized molybdenum oxide nanozyme for specific colorimetric monitoring of xanthine oxidase activity.

Xanthine-functionalized molybdenum oxide nanodots (X-MoO3-x NDs) with peroxidase (POD)-like activity were developed for selective, sensitive, and facile colorimetric quantification of xanthine oxidase (XO). Xanthine functionalization can not only be favorable for the successful nanozyme preparation, but also for the specific recognition of XO as well as the simultaneous generation of hydrogen peroxide, which was subsequently transformed into hydroxyl radical to oxidize the chromogenic reagent based on the POD-like catalysis. Under the optimized conditions, the colorimetric biosensing platform was established for XO assay without addition of further substrates, showing good linearity relationship between absorbance difference (ΔA) and XO concentrations in the range 0.05-0.5 U/mL (R2 = 0.998) with a limit of detection (LOD) of 0.019 U/mL. The quantification of XO occurs in 25 min, which is superior to the previously reported and commercial XO assays. The proposed method has been successfully used in the assay of human serum samples, showing its high potential in the field of clinical monitoring.

Assessment of Bitterness in Non-Charged Pharmaceuticals with a Taste Sensor: A Study on Substances with Xanthine Scaffold and Allopurinol.

Taste sensors with an allostery approach have been studied to detect non-charged bitter substances, such as xanthine derivatives, used in foods (e.g., caffeine) or pharmaceuticals (e.g., etofylline). In this study, the authors modified a taste sensor with 3-bromo-2,6-dihydroxybenzoic acid and used it in conjunction with sensory tests to assess the bitterness of non-charged pharmaceuticals with xanthine scaffolds (i.e., acefylline and doxofylline), as well as allopurinol, an analogue of hypoxanthine. The results show that the sensor was able to differentiate between different levels of sample bitterness. For instance, when assessing a 30 mM sample solution, the sensor response to acefylline was 34.24 mV, which corresponded to the highest level of bitterness (τ = 3.50), while the response to allopurinol was lowest at 2.72 mV, corresponding to relatively weaker bitterness (τ = 0.50). Additionally, this study extended the application of the sensor to detect pentoxifylline, an active pharmaceutical ingredient in pediatric medicines. These results underscore the taste sensor's value as an additional tool for early-stage assessment and prediction of bitterness in non-charged pharmaceuticals.

Structure-Activity Relationship Studies in a Series of Xanthine Inhibitors of SLACK Potassium Channels.

Gain-of-function mutations in the KCNT1 gene, which encodes the sodium-activated potassium channel known as SLACK, are associated with the rare but devastating developmental and epileptic encephalopathy known as epilepsy of infancy with migrating focal seizures (EIMFS). The design of small molecule inhibitors of SLACK channels represents a potential therapeutic approach to the treatment of EIMFS, other childhood epilepsies, and developmental disorders. Herein, we describe a hit optimization effort centered on a xanthine SLACK inhibitor (8) discovered via a high-throughput screen. Across three distinct regions of the chemotype, we synthesized 58 new analogs and tested each one in a whole-cell automated patch-clamp assay to develop structure-activity relationships for inhibition of SLACK channels. We further evaluated selected analogs for their selectivity versus a variety of other ion channels and for their activity versus clinically relevant SLACK mutants. Selectivity within the series was quite good, including versus hERG. Analog 80 (VU0948578) was a potent inhibitor of WT, A934T, and G288S SLACK, with IC50 values between 0.59 and 0.71 µM across these variants. VU0948578 represents a useful in vitro tool compound from a chemotype that is distinct from previously reported small molecule inhibitors of SLACK channels.

Multi-omics identify xanthine as a pro-survival metabolite for nematodes with mitochondrial dysfunction.

Aberrant mitochondrial function contributes to the pathogenesis of various metabolic and chronic disorders. Inhibition of insulin/IGF-1 signaling (IIS) represents a promising avenue for the treatment of mitochondrial diseases, although many of the molecular mechanisms underlying this beneficial effect remain elusive. Using an unbiased multi-omics approach, we report here that IIS inhibition reduces protein synthesis and favors catabolism in mitochondrial deficient Caenorhabditis elegans We unveil that the lifespan extension does not occur through the restoration of mitochondrial respiration, but as a consequence of an ATP-saving metabolic rewiring that is associated with an evolutionarily conserved phosphoproteome landscape. Furthermore, we identify xanthine accumulation as a prominent downstream metabolic output of IIS inhibition. We provide evidence that supplementation of FDA-approved xanthine derivatives is sufficient to promote fitness and survival of nematodes carrying mitochondrial lesions. Together, our data describe previously unknown molecular components of a metabolic network that can extend the lifespan of short-lived mitochondrial mutant animals.

Signal-enhanced multi-core fiber-based WaveFlex biosensor for ultra-sensitive xanthine detection.

In this work, we introduce a novel multimode fiber (MMF) - seven core fiber (SCF) - MMF (MCM) optical fiber biosensor, also known as the WaveFlex biosensor (plasma wave assisted fiber biosensor), based on localized surface plasmon resonance (LSPR) for qualitative detection of xanthine. Xanthine is a purine base widely distributed in human blood and tissues, and commonly used as an indicator for various disease detections. The MCM sensor incorporates a tapered optical fiber structure, fabricated using the combiner manufacturing system (CMS), and is designed with SCF and MMF. By effectively harnessing LSPR, the sensor boosts the attachment points of biomolecules on the probe surface through immobilized tungsten disulfide (WS2)-thin layers, gold nanoparticles (AuNPs), and carbon nitride quantum dots (C3N-QDs). The functionalization of xanthine oxidase (XO) on the sensing probe further enhances the sensor's specificity. The proposed WaveFlex biosensor exhibits a remarkable sensitivity of 3.2 nm/mM and a low detection limit of 96.75 µM within the linear detection range of 100 - 900 µM. Moreover, the sensor probe demonstrates excellent reusability, reproducibility, stability, and selectivity. With its sensitivity, biocompatibility, and immense potential for detecting human serum and fish products, this WaveFlex biosensor presents a promising platform for future applications.

Simple assay for quantifying xanthine oxidase activity.

This paper reports a sensitive method for assaying xanthine oxidase (XO) enzyme activity. XO produces hydrogen peroxide (H2O2) and superoxide anion radicals (O2•-), promoting the development of oxidative stress-related diseases, and is inhibited by various plant extracts. XO activity is quantified by incubating enzyme samples with an appropriate xanthine concentration as the substrate. The proposed method requires XO activity to be quantified based on H2O2 generation using a 3,3',5,5'-tetramethylbenzidine (TMB)-H2O2 system catalyzed by cupric ions. After a 30-min incubation at 37 °C, sufficient cupric ion and TMB amounts are added. The assay produces optical signals that can be visually recognized or detected with a UV-visible spectrometer. A direct correlation was found between XO activity and the absorbance at 450 nm of the resulting di-imine (dication) yellow product. The proposed method uses sodium azide to prevent catalase enzyme interference. The new assay's function was confirmed using the TMB-XO assay and a Bland-Altman plot. The resulting correlation coefficient was 0.9976. The innovative assay was relatively precise and comparable to the comparison protocols. In conclusion, the presented method is very efficient at measuring XO activity.

Purine DNA Constructs Designed to Expand the Genetic Code: Functionalization, Impact of Ionic Forms, and Molecular Recognition of 7-Deazaxanthine-7-Deazapurine-2,6-diamine Base Pairs and Their Purine Counterparts.

Purine DNA represents an alternative pairing system formed by two purines in the base pair with the recognition elements of Watson-Crick DNA. Base functionalization of 7-deaza-2'-deoxyxanthosine with ethynyl and octadiynyl residues led to clickable side chain derivatives with short and long linker arms. As complementary bases, purine-2,6-diamine or 7-deazapurine-2,6-diamine 2'-deoxyribonucleosides were used. 7-Deaza-7-iodo-2'-deoxyxanthosine served as a starting material for Sonogashira cross-coupling and the p-nitrophenylethyl group for base protection. Phosphoramidite building blocks for DNA synthesis were prepared. Oligonucleotides containing single modifications or runs of three purine base pairs embedded in 12-mer Watson-Crick DNA were synthesized and hybridized with complementary strands with purine- or 7-deazapurine-2,6-diamine located opposite to the xanthine derivatives. The stability of base pairs was evaluated in a comparative study on the basis of DNA melting experiments and Tm values. As 7-deazaxanthine and xanthine nucleosides form anionic forms at neutral pH, duplex stability became pK-dependent, and the system with 7-deazapurine displayed a significant higher stability as that containing xanthine. Alkynyl side chains are well accommodated in the purine-purine helix. Click adducts with pyrene showed that short linker arms destabilize duplexes, whereas long linkers increase duplex stability. CD and fluorescence measurements provide further insights into purine-purine base pairing.

A ratiometric SERS sensor with one signal probe for ultrasensitive and quantitative monitoring of serum xanthine.

Xanthine can be converted into uric acid, and a high concentration of xanthine in the human body can cause many diseases. Therefore, it is important to develop a sensitive, simple, and reliable approach for measuring xanthine in biological liquids. Hence, a ratiometric surface-enhanced Raman spectroscopy (SERS) sensing strategy with one signal probe was exploited for reliable, sensitive, and quantitative monitoring of serum xanthine. 3-Mercaptophenylboronic acid (3-MPBA) was used as a typical reference with a Raman peak at 996 cm-1. First, 3-MPBA was bound to gold nanoflowers@silica (GNFs@Si) through Au-S bonds. Xanthine oxidase (XOD) catalyzed the oxidation of xanthine into H2O2 on GNFs@Si. Afterward, the obtained H2O2 further reduced 3-MPBA to 3-hydroxythiophenol (3-HTP) accompanied by the emergence of a new Raman peak at 883 cm-1. Meanwhile, the Raman intensity at 996 cm-1 remained constant. Therefore, the ratio of I883/I996 increased with the increasing of xanthine concentration, thus realizing quantitative detection of xanthine. As a result, a ratiometric SERS sensor for the detection of xanthine was proposed with a detection limit of 5.7 nM for xanthine. The novel ratiometric SERS sensor provides a new direction for analyzing other biomolecules with high sensitivity and reliability.

Investigation of the Inhibition Mechanism of Xanthine Oxidoreductase by Oxipurinol: A Computational Study.

Xanthine oxidoreductase (XOR) is an enzyme found in various organisms. It converts hypoxanthine to xanthine and urate, which are crucial steps in purine elimination in humans. Elevated uric acid levels can lead to conditions like gout and hyperuricemia. Therefore, there is significant interest in developing drugs that target XOR for treating these conditions and other diseases. Oxipurinol, an analogue of xanthine, is a well-known inhibitor of XOR. Crystallographic studies have revealed that oxipurinol directly binds to the molybdenum cofactor (MoCo) in XOR. However, the precise details of the inhibition mechanism are still unclear, which would be valuable for designing more effective drugs with similar inhibitory functions. In this study, molecular dynamics and quantum mechanics/molecular mechanics calculations are employed to investigate the inhibition mechanism of XOR by oxipurinol. The study examines the structural and dynamic effects of oxipurinol on the pre-catalytic structure of the metabolite-bound system. Our results provide insights on the reaction mechanism catalyzed by the MoCo center in the active site, which aligns well with experimental findings. Furthermore, the results provide insights into the residues surrounding the active site and propose an alternative mechanism for developing alternative covalent inhibitors.

A liquid chromatography-tandem mass spectrometry method for comprehensive determination of metabolites in the purine pathway of rat plasma and its application in anti-gout effects of Lycium ruthenicum Murr.

It has been proved that purine metabolites are implicated in various biological syndromes and disorders. Therefore, the realization of panoramic detection of purine metabolites will be of great significance to the pathogenesis of purine metabolic disorders. In the present study, an ultra-high performance liquid chromatography with tandem mass spectrometry method was developed for the comprehensive quantification of purine metabolites in rat plasma. The 17 purine metabolites were separated and quantified in the short running time of 15 min. The proposed method was strictly validated by applying SeraSub solution as a matrix and proved to be linear (R2 ≥ 0.9944), accurate (the recoveries of all analytes ranged from 85.3% to 103.0%, with relative standard deviation values ≤ 9.3%), and precise (the intra- and inter-day precisions were less than 10.8% and 12.4%, respectively). The method was then successfully applied to the qualification of the endogenous purine metabolites in acute gouty arthritis rats, as well as colchicine and anthocyanin-intervened rats. Results showed that uric acid, xanthine, hypoxanthine, and xanthine were considered the key factors of acute gouty arthritis. The established method and measurement of purines in rat plasma might help the investigation of the action mechanisms between purine disorders and related diseases.

The role of xanthine dioxygenase in the biosynthetic pathway of 2-aza-8-oxohypoxanthine of Lepista sordida.

2-Azahypoxanthine (AHX) and 2-aza-8-oxohypoxanthine (AOH), discovered as causal substances of fairy rings are known to be endogenous in the fairy ring-forming Lepista sordida. In this study, we showed that xanthine dioxygenase, an a-ketoglutarate-dependent dioxygenase, might catalyze the conversion of AHX to AOH in the fungus. Furthermore, this enzyme is the first reported molybdopterin-independent protein of hypoxanthine metabolism.

Declaration: Novel SLC3A1 mutation in a cystinuria patient with xanthine stones: a case report.

BACKGROUND: Cystinuria and xanthinuria are both rare genetic diseases involving urinary calculi. However, cases combining these two disorders have not yet been reported. CASE PRESENTATION: In this study, we report a case of cystinuria with xanthine stones and hyperuricemia. The 23-year-old male patient was diagnosed with kidney and ureteral stones, solitary functioning kidney and hyperuricemia after admission to the hospital. The stones were removed by surgery and found to be composed of xanthine. CONCLUSION: Genetic testing by next-generation sequencing technology showed that the patient carried the homozygous nonsense mutation c.1113 C> A (p.Tyr371*) in the SLC3A1 gene, which was judged to be a functionally pathogenic variant. Sanger sequencing revealed that the patient's parents carried this heterozygous mutation, which is a pathogenic variant that can cause cystinuria. The 24-h urine metabolism analysis showed that the cystine content was 644 mg (<320 mg/24 h), indicating that the patient had cystinuria, consistent with the genetic test results. This case shows that cystinuria and xanthine stones can occur simultaneously, and provides evidence of a possible connection between the two conditions. Furthermore, our findings demonstrate the potential value of genetic testing using next-generation sequencing to effectively assist in the clinical diagnosis and treatment of patients with urinary calculi.

Candidate causative variant for xanthinuria in a Domestic Shorthair cat.

Xanthinuria is a clinically significant form of urolithiasis in cats with poor clinical outcomes and limited treatment options. In humans, xanthinuria has an autosomal recessive mode of inheritance, with variants in xanthine dehydrogenase (XDH) and molybdenum cofactor sulfurase (MOCOS) responsible for cases. While causative genetic variants have not been identified in the domestic cat, a recessive mode of inheritance has been suggested. DNA was extracted from EDTA-stabilised blood obtained from a Domestic Shorthair cat with clinically confirmed xanthinuria. Whole-genome sequencing and variant assessment in XDH and MOCOS identified XDH:c.2042C>T (XDH:p.(A681V)) as a candidate causative variant for xanthinuria in this cat. The variant is located in a highly conserved part of the molybdenum-pterin co-factor domain, responsible for catalysing the hydroxylation of hypoxanthine to xanthine and uric acid. Variants in this domain of XDH have been shown to disrupt enzyme function and to cause xanthinuria in other species. When assessed in the wider cat population, the variant had an allele frequency of 15.8%, with 0.9% of the animals assessed homozygous for the alternative allele. Cats diagnosed with xanthinuria should be tested for this variant to validate its clinical relevance in the wider population.

Insights into xanthine riboswitch structure and metal ion-mediated ligand recognition.

Riboswitches are conserved functional domains in mRNA that mostly exist in bacteria. They regulate gene expression in response to varying concentrations of metabolites or metal ions. Recently, the NMT1 RNA motif has been identified to selectively bind xanthine and uric acid, respectively, both are involved in the metabolic pathway of purine degradation. Here, we report a crystal structure of this RNA bound to xanthine. Overall, the riboswitch exhibits a rod-like, continuously stacked fold composed of three stems and two internal junctions. The binding-pocket is determined by the highly conserved junctional sequence J1 between stem P1 and P2a, and engages a long-distance Watson-Crick base pair to junction J2. Xanthine inserts between a G-U pair from the major groove side and is sandwiched between base triples. Strikingly, a Mg2+ ion is inner-sphere coordinated to O6 of xanthine and a non-bridging oxygen of a backbone phosphate. Two further hydrated Mg2+ ions participate in extensive interactions between xanthine and the pocket. Our structure model is verified by ligand binding analysis to selected riboswitch mutants using isothermal titration calorimetry, and by fluorescence spectroscopic analysis of RNA folding using 2-aminopurine-modified variants. Together, our study highlights the principles of metal ion-mediated ligand recognition by the xanthine riboswitch.

Locking mixed-lineage kinase domain-like protein in its auto-inhibited state prevents necroptosis.

As an alternative pathway of controlled cell death, necroptosis can be triggered by tumor necrosis factor via the kinases RIPK1/RIPK3 and the effector protein mixed-lineage kinase domain-like protein (MLKL). Upon activation, MLKL oligomerizes and integrates into the plasma membrane via its executioner domain. Here, we present the X-ray and NMR costructures of the human MLKL executioner domain covalently bound via Cys86 to a xanthine class inhibitor. The structures reveal that the compound stabilizes the interaction between the auto-inhibitory brace helix α6 and the four-helix bundle by stacking to Phe148. An NMR-based functional assay observing the conformation of this helix showed that the F148A mutant is unresponsive to the compound, providing further evidence for the importance of this interaction. Real-time and diffusion NMR studies demonstrate that xanthine derivatives inhibit MLKL oligomerization. Finally, we show that the other well-known MLKL inhibitor Necrosulfonamide, which also covalently modifies Cys86, must employ a different mode of action.

Antiplatelet Effects of Selected Xanthine-Based Adenosine A2A and A2B Receptor Antagonists Determined in Rat Blood.

The platelet aggregation inhibitory activity of selected xanthine-based adenosine A2A and A2B receptor antagonists was investigated, and attempts were made to explain the observed effects. The selective A2B receptor antagonist PSB-603 and the A2A receptor antagonist TB-42 inhibited platelet aggregation induced by collagen or ADP. In addition to adenosine receptor blockade, the compounds were found to act as moderately potent non-selective inhibitors of phosphodiesterases (PDEs). TB-42 showed the highest inhibitory activity against PDE3A along with moderate activity against PDE2A and PDE5A. The antiplatelet activity of PSB-603 and TB-42 may be due to inhibition of PDEs, which induces an increase in cAMP and/or cGMP concentrations in platelets. The xanthine-based adenosine receptor antagonists were found to be non-cytotoxic for platelets. Some of the compounds showed anti-oxidative properties reducing lipid peroxidation. These results may provide a basis for the future development of multi-target xanthine derivatives for the treatment of inflammation and atherosclerosis and the prevention of heart infarction and stroke.

Simultaneous Determination of Xanthine and Hypoxanthine Using Polyglycine/rGO-Modified Glassy Carbon Electrode.

A novel electrochemical sensor was developed for selective and sensitive determination of xanthine (XT) and hypoxanthine (HX) based on polyglycine (p-Gly) and reduced graphene oxide (rGO) modified glassy carbon electrode (GCE). A mixed dispersion of 7 µL of 5 mM glycine and 1 mg/mL GO was dropped on GCE for the fabrication of p-Gly/rGO/GCE, followed by cyclic voltammetric sweeping in 0.1 M phosphate buffer solution within -0.45~1.85 V at a scanning rate of 100 mV·s-1. The morphological and electrochemical features of p-Gly/rGO/GCE were investigated by scanning electron microscopy and cyclic voltammetry. Under optimal conditions, the linear relationship was acquired for the simultaneous determination of XT and HX in 1-100 µM. The preparation of the electrode was simple and efficient. Additionally, the sensor combined the excellent conductivity of rGO and the polymerization of Gly, demonstrating satisfying simultaneous sensing performance to both XT and HX.