Green manufacturing of a hypoxanthine enzyme sensor for fish freshness based on modified nitrocellulose surface with chito-oligosaccharide.

Hypoxanthine (Hx), produced by adenosine triphosphate (ATP) metabolism, is a valuable indicator that determines the quality and degradation status of meat products and is also an important biochemical marker to certain diseases such as gout. The rapid emergence of paper-based enzyme biosensors has already revolutionized its on-site determination. But it is still limited by the complex patterning and fabrication, unstable enzyme and uneven coloration. This work aims to develop an eco-friendly method to construct engineered paper microfluidic, which seeks to produce reaction and non-reaction zones without any patterning procedure. Chito-oligosaccharide (COS), derived from shrimp shells, was used to modify nitrocellulose membranes and immobilize xanthine oxidase (XOD) and chromogenic agent of nitro blue tetrazolium chloride (NBT). After modification, micro fluids could converge into the modification area and Hx could be detected by XOD-catalyzed conversion. Due to the positively charged cationic basic properties of COS, the enzyme storage stability and the color homogeneity could be greatly strengthened through the electrostatic attraction between COS and XOD and formazan product. The detection limit (LOD) is 2.30 µM; the linear range is 0.05-0.35 mM; the complete test time can be as short as 5 min. The COS-based biosensor shows high specificity and can be used directly for Hx in complex samples such as fish and shrimp samples, and different broths. This biosensor is eco-friendly, nontechnical, economical and therefore a compelling platform for on-site or home-based detection of food freshness.

Brushite nanoparticles based electrochemical sensor for detection of uric acid, xanthine, hypoxanthine and caffeine.

The research in the field of biosensors has recently been focused on the design and development of functional electrode materials that can respond to changes in their biochemical environment. Here, we report the synthesis of dicalcium phosphate dihydrate (DCPD), also known as brushite (CaHPO4·2H2O) by soft chemical method and its application for electrochemical sensing of four different analytes. Phase purity, structure, chemical composition and surface morphology of the synthesized nanoparticles have been investigated using powder XRD, FTIR, SEM, XPS and HRTEM methods. Electrochemical sensor was prepared by modifying GCE with brushite and the modified electrodes were successfully used for either independent or simultaneous determination of uric acid, xanthine, hypoxanthine and caffeine in their mixture. The brushite/GCE exhibited four strong well-defined separate peaks corresponding to the oxidation of UA, XN, HXN and CF in phosphate buffer saline (PBS) at pH 7.4. The fabricated electrode showed low detection limits (S/N = 3) of 0.576, 1.0, 0.076 and 1.26 µM for UA, XN, HXN and CF respectively. Practical application of the fabricated electrode has been demonstrated by determining UA, XN, HXN and CF in human urine and coffee samples by direct method. The brushite offers scope for fabrication of sensor systems for implantable medical applications.

AgCu@CuO aerogels with peroxidase-like activities and photoelectric responses for sensitive biosensing.

Photoelectrochemical (PEC) enzymatic biosensors integrate the excellent selectivity of enzymes and high sensitivity of PEC bioanalysis, but the drawbacks such as high cost, poor stability, and tedious immobilization of natural enzymes on photoelectrodes severely suppress their applications. AgCu@CuO aerogel-based photoelectrode materials with both remarkable enzyme-like activities and outstanding photoelectric properties were innovatively designed and synthesized to evaluate the activity of xanthine oxidase with a wide linear detection range and a low limit of detection.

Synthesis and biological evaluation of all possible inosine-mixed cyclic dinucleotides that activate different hSTING variants.

Cyclic dinucleotides (CDNs) could activate stimulator of interferon genes (STING) protein to produce type I interferon and other pro-inflammation cytokines in mammalian cells. To explore new types of potentially efficient STING activators targeting all five major hSTING variants (WT, R232H, HAQ, AQ and R293Q), we here reported the synthesis of a total of 19 inosine-containing CDNs based on the combinations of hypoxanthine with four natural bases (A, G, C and U) and three phosphodiester linkage backbones (3'-3', 2'-3', 2'-2'). The IFN-ß induction results showed that all of the 2'-3' and 2'-2' CDNs linked by inosine and purine nucleosides favored the stacking interaction with Y167 and R238 residues of hSTING protein, and several CDNs constructed by hypoxanthine and pyrimidine like c[I(2',5')U(2',5')] could also activate all five hSTING variants. The molecular dynamic simulation and the isothermal titration calorimetric (ITC) assay further demonstrated the potential of cAIMP isomers with 2'-5' phosphate to form the hydrogen binding with R232 and R238 residues of hSTING in an entropically driven manner compared to cGAMP isomers. It would be promising to exploit novel inosine-mixed CDNs as activators of hSTING variants in immune therapy.

Frequency and hydrogen bonding of nucleobase homopairs in small molecule crystals.

We used the high resolution and accuracy of the Cambridge Structural Database (CSD) to provide detailed information regarding base pairing interactions of selected nucleobases. We searched for base pairs in which nucleobases interact with each other through two or more hydrogen bonds and form more or less planar structures. The investigated compounds were either free forms or derivatives of adenine, guanine, hypoxanthine, thymine, uracil and cytosine. We divided our findings into categories including types of pairs, protonation patterns and whether they are formed by free bases or substituted ones. We found base pair types that are exclusive to small molecule crystal structures, some that can be found only in RNA containing crystal structures and many that are native to both environments. With a few exceptions, nucleobase protonation generally followed a standard pattern governed by pKa values. The lengths of hydrogen bonds did not depend on whether the nucleobases forming a base pair were charged or not. The reasons why particular nucleobases formed base pairs in a certain way varied significantly.

Novel electrochemical method to evaluate the antioxidant capacity of infusions and beverages, based on in situ formation of free superoxide radicals.

This work reports a new method to evaluate the antioxidant capacity of infusions and beverages, based on superoxide radicals. Radicals produced by the enzymatic reaction between acetylcholinesterase and hypoxanthine oxidized antioxidant molecules present in commercially available samples or standard solutions, which was monitored by means of cyclic voltammetry using a carbon paste electrode. The Trolox equivalent antioxidant capacity (TEAC) of red wine, coffee and green tea determined using this method were: (1.20 ± 0.06), (0.90 ± 0.02), and (0.65 ± 0.02), respectively. This method suggested TEACred wine > TEACcoffee > TEACgreen tea, which is the same as DPPH, spectrophotometric method. However, the electrochemical one proposed here is rapid and simple.

Discovery and evaluation of new compounds targeting ribosomal protein S1 in antibiotic-resistant Mycobacterium Tuberculosis.

The emergence of antibiotic-resistant Mycobacterium Tuberculosis (Mtb) infections compels new treatment strategies, of which targeting trans-translation is promising. During the trans-translation process, the ribosomal protein S1 (RpsA) plays a key role, and the Ala438 mutant is related to pyrazinamide (PZA) resistance, which shows its effects after being hydrolysed to pyrazinoic acid (POA). In this study, based on the structure of the RpsA C-terminal domain (RpsA-CTD) and POA complex, new compounds were designed. After being synthesized, the compounds were tested in vitro with saturation transfer difference (STD), fluorescence quenching titration (FQT) and chemical shift perturbation (CSP) experiments. Finally, six of the 17 new compounds have high affinity for both RpsA-CTD and its Ala438 deletion mutant. The active compounds provide new choices for targeting trans-translation in Mtb, and the analysis of the structure-activity relationships will be helpful for further structural modifications based on derivatives of 2-((hypoxanthine-2-yl)thio)acetic acid and 2-((5-hydroxylflavone-7-yl)oxy)acetamide.

Coupling of cyclo-l-Trp-l-Trp with Hypoxanthine Increases the Structure Diversity of Guanitrypmycins.

The cyclo-l-Trp-l-Trp (cWW, 1) tailoring P450 GutD2774 from Streptomyces lavendulae was characterized by expression in Streptomyces coelicolor, precursor feeding and enzyme assays. GutD2774 catalyzes mainly the transfer of hypoxanthine to C2 and C3 of the indole ring of 1. cWW adducts with guanine were detected as minor products. An orthologous cluster was identified in Streptomyces xanthophaeus. These results expand the spectrum of cyclodipeptide derivatives by involvement of an additional nucleobase and identification of new coupling patterns.

Nucleobase synthesis in interstellar ices.

The synthesis of nucleobases in natural environments, especially in interstellar molecular clouds, is the focus of a long-standing debate regarding prebiotic chemical evolution. Here we report the simultaneous detection of all three pyrimidine (cytosine, uracil and thymine) and three purine nucleobases (adenine, xanthine and hypoxanthine) in interstellar ice analogues composed of simple molecules including H2O, CO, NH3 and CH3OH after exposure to ultraviolet photons followed by thermal processes, that is, in conditions that simulate the chemical processes accompanying star formation from molecular clouds. Photolysis of primitive gas molecules at 10 K might be one of the key steps in the production of nucleobases. The present results strongly suggest that the evolution from molecular clouds to stars and planets provides a suitable environment for nucleobase synthesis in space.

In-situ reduction of Ag+ on black phosphorene and its NH2-MWCNT nanohybrid with high stability and dispersibility as nanozyme sensor for three ATP metabolites.

The environmental stability, water-processibility and life-span of black phosphorene (BP) severely limit the application of its electronic devices in aqueous system containing oxygen. We reported the controllable preparation of in-situ reduction and deposition of silver nanoparticles on the BP surface and its amino-functionalized multi-walled carbon nanotubes (NH2-MWCNT) nanocomposite. With the addition of both NH2-MWCNT and Ag+, the BP-based nanocomposite was prepared by ultrasonic-assisted liquid-phase exfoliation and was dispersed in carboxymethyl cellulose sodium (CMC) aqueous solution. The morphology, microstructure, and electrochemical properties of the nanohybrid were characterized. NH2-MWCNT-BP-AgNPs showed high environmental stability, good water-processibility, satisfactory life-spans, superior electrocatalytic capacity with enzyme-like kinetic characteristics. The nanohybrid was applied as electrochemical sensors for single/simultaneous analysis of uric acid (UA), xanthine (XT) and hypoxanthine (HX). Excellent voltammetric responses for simultaneous determination in linear ranges of 0.1-800 µM with a limit of detection (LOD) of 0.052 µM for UA, 0.5-680 µM with a LOD of 0.021 µM for XT, and 0.7-320 µM with a LOD of 0.025 µM for HX under optimal conditions. Besides, the developed nanozyme sensor was employed for simultaneous voltammetric analysis of UA, XT and HX in real samples with acceptable recoveries. This work will provide theoretical guidance and experimental support for the preparation and application of two-dimensional materials, nanozymes and sensing devices.

Spectroscopic and computational exploration of hypoxanthine riboside interacting with plasma albumin.

Hypoxanthine riboside (HXR) is a nucleoside essential for wobble base pairs to translate the genetic code. In this work, an absorption and luminescence study showed that HXR and human serum albumin (HSA) formed a new complex through hydrogen bonds and van der Waals forces at ground state. Fluorescence probe experiments indicated that HXR entered the first subdomain of domain II in HSA and was fixed by amino acid residues in site I defined by Sudlow, and after competing with a known site marker. The recognition interaction featured negative ΔHÏ´ , ΔSÏ´ and ΔGÏ´ thermodynamic parameters. Fluorescence and circular dichroism spectra described the polarity of residues and α-helix and ß-strand content changed because of HXR binding. The most rational structure for the HXR-HSA complex was recommended by the molecular docking method, in which the binding location, molecular orientation, adjacent amino acid residues, and hydrogen bonds were included. In addition, the influence of ß-cyclodextrin and some essential metal ions on the balance of the HSA-HXR system interaction was measured. The study gained comprehensive information on the transportation mechanism for HXR in blood, and was of great significance in understanding the theory of HXR biotransformation and in discussing its clinical in vivo half-life.

Development of a highly sensitive xanthine oxidase-based biosensor for the determination of antioxidant capacity in Amazonian fruit samples.

The paper describes the development of an amperometric biosensor using Prussian Blue (PB) modified electrodes containing xanthine oxidase (XOD). The enzyme is immobilized by photo-polymerization into an azide-unit pendant water-soluble photopolymer (PVA-AWP). The parameters of the fabrication of the biosensor, XOD:PVA/AWP ratio, crosslinking irradiation time, and XOD charge, were optimized. Operational conditions for electrode preparation were defined as 1:2 ratio of XOD:PVA/AWP; exposure time to neon light of 30  min; pH = 7.5  at room temperature and enzymatic charge of 8 mU per electrode. The biosensors showed stable, fast, simple, selective, cost-effective and sensitive (-2.72E-8  A mol L-1), with a good linear range (1.0-75  µmol L-1), and respectively detection and quantification limits for antioxidants of 2.17, and 7.15  µmol L-1. The applicability of this biosensor was demonstrated by in vitro analysis of gallic acid as standard antioxidant and Amazonian fruits as natural sources.

Base-Pairing Properties of Double-Headed Nucleotides.

Nucleotides that contain two nucleobases (double-headed nucleotides) have the potential to condense the information of two separate nucleotides into one. This presupposes that both bases must successfully pair with a cognate strand. Here, double-headed nucleotides that feature cytosine, guanine, thymine, adenine, hypoxanthine, and diaminopurine linked to the C2'-position of an arabinose scaffold were developed and examined in full detail. These monomeric units were efficiently prepared by convergent synthesis and incorporated into DNA oligonucleotides by means of the automated phosphoramidite method. Their pairing efficiency was assessed by UV-based melting-temperature analysis in several contexts and extensive molecular dynamics studies. Altogether, the results show that these double-headed nucleotides have a well-defined structure and invariably behave as functional dinucleotide mimics in DNA duplexes.

The influence of Cu+ binding to hypoxanthine on stabilization of mismatches involving hypoxanthine and DNA bases: a DFT study.

In the present work, the influence of Cu+ binding to N3- and N7-positions of hypoxanthine on energetic, geometrical and topological properties of hypoxanthine-guanine, hypoxanthine-adenine, hypoxanthine-cytosine, hypoxanthine-thymine and hypoxanthine-hypoxanthine mismatches is theoretically investigated. The calculations, in gas phase, are performed at B3LYP/6-311++G(3df,3pd) level of theory. Unlike the other mispairs, Cu+ binding to N3-position of hypoxanthine causes the proton transfer process from enol form of hypoxanthine to imino forms of adenine and cytosine. This process also occurs in all mismatches having enol form of hypoxanthine when Cu+ binds to N7-position of hypoxanthine. The mismatches are stabilized by hydrogen bonds. The influence of Cu+ on hydrogen bonds is also examined by atoms in molecules (AIM) and natural bond orbital (NBO) analyses. Communicated by Ramaswamy H. Sarma.

Microtubule-Targeting 7-Deazahypoxanthines Derived from Marine Alkaloid Rigidins: Exploration of the N3 and N9 Positions and Interaction with Multidrug-Resistance Proteins.

Our laboratories have been investigating synthetic analogues of marine alkaloid rigidins that possess promising anticancer activities. These analogues, based on the 7-deazahypoxanthine skeleton, are available in one- or two-step synthetic sequences and exert cytotoxicity by disrupting microtubule dynamics in cancer cells. In the present work we extended the available structure-activity relationship (SAR) data to N3- and N9-substituted derivatives. Although N3 substitution results in loss of activity, the N9-substituted compounds retain nanomolar antiproliferative activities and the anti-tubulin mode of action of the original unsubstituted compounds. Furthermore, our results also demonstrate that multidrug-resistance (MDR) proteins do not confer resistance to both N9-unsubstituted and -substituted compounds. It was found that sublines overexpressing ABCG2, ABCC1, and ABCB1 proteins are as responsive to the rigidin analogues as their parental cell lines. Thus, the study reported herein provides further impetus to investigate the rigidin-inspired 7-deazahypoxanthines as promising anticancer agents.

Structural explanation for the tunable substrate specificity of an E. coli nucleoside hydrolase: insights from molecular dynamics simulations.

Parasitic protozoa rely on nucleoside hydrolases that play key roles in the purine salvage pathway by catalyzing the hydrolytic cleavage of the N-glycosidic bond that connects nucleobases to ribose sugars. Cytidine-uridine nucleoside hydrolase (CU-NH) is generally specific toward pyrimidine nucleosides; however, previous work has shown that replacing two active site residues with Tyr, specifically the Thr223Tyr and Gln227Tyr mutations, allows CU-NH to process inosine. The current study uses molecular dynamics (MD) simulations to gain atomic-level insight into the activity of wild-type and mutant E. coli CU-NH toward inosine. By examining systems that differ in the identity and protonation states of active site catalytic residues, key enzyme-substrate interactions that dictate the substrate specificity of CU-NH are identified. Regardless of the wild-type or mutant CU-NH considered, our calculations suggest that inosine binding is facilitated by interactions of the ribose moiety with active site residues and Ca2+, and π-interactions between two His residues (His82 and His239) and the nucleobase. However, the lack of observed activity toward inosine for wild-type CU-NH is explained by no residue being correctly aligned to stabilize the departing nucleobase. In contrast, a hydrogen-bonding network between hypoxanthine and a newly identified general acid (Asp15) is present when the two Tyr mutations are engineered into the active site. Investigation of the single CU-NH mutants reveals that this hydrogen-bonding network is only maintained when both Tyr mutations are present due to a π-interaction between the residues. These results rationalize previous experiments that show the single Tyr mutants are unable to efficiently hydrolyze inosine and explain how the Tyr residues work synergistically in the double mutant to stabilize the nucleobase leaving group during hydrolysis. Overall, our simulations provide a structural explanation for the substrate specificity of nucleoside hydrolases, which may be used to rationally develop new treatments for kinetoplastid diseases.

Effect of corn supplementation on purine derivatives and rumen fermentation in sheep fed PKC and urea-treated rice straw.

This study investigated the effect of different levels of corn supplementation as energy source into palm kernel cake-urea-treated rice straw basal diet on urinary excretion of purine derivatives, nitrogen utilization, rumen fermentation, and rumen microorganism populations. Twenty-seven Dorper lambs were randomly assigned to three treatment groups and kept in individual pens for a 120-day period. The animals were subjected to the dietary treatments as follows: T1: 75.3% PKC + 0% corn, T2: 70.3% PKC + 5% corn, and T3: 65.3% PKC + 10% corn. Hypoxanthine and uric acid excretion level were recorded similarly in lambs supplemented with corn. The microbial N yield and butyrate level was higher in corn-supplemented group, but fecal N excretion, T3 has the lowest level than other groups. Lambs fed T3 had a greater rumen protozoa population while the number of R. flavefaciens was recorded highest in T2. No significant differences were observed for total bacteria, F. succinogenes, R. albus, and methanogen population among all treatment. Based on these results, T3 could be fed to lambs without deleterious effect on the VFA and N balance.

Crystal structure of Escherichia coli purine nucleoside phosphorylase in complex with 7-deazahypoxanthine.

Purine nucleoside phosphorylases (EC 2.4.2.1; PNPs) reversibly catalyze the phosphorolytic cleavage of glycosidic bonds in purine nucleosides to generate ribose 1-phosphate and a free purine base, and are key enzymes in the salvage pathway of purine biosynthesis. They also catalyze the transfer of pentosyl groups between purine bases (the transglycosylation reaction) and are widely used for the synthesis of biologically important analogues of natural nucleosides, including a number of anticancer and antiviral drugs. Potent inhibitors of PNPs are used in chemotherapeutic applications. The detailed study of the binding of purine bases and their derivatives in the active site of PNPs is of particular interest in order to understand the mechanism of enzyme action and for the development of new enzyme inhibitors. Here, it is shown that 7-deazahypoxanthine (7DHX) is a noncompetitive inhibitor of the phosphorolysis of inosine by recombinant Escherichia coli PNP (EcPNP) with an inhibition constant Ki of 0.13 mM. A crystal of EcPNP in complex with 7DHX was obtained in microgravity by the counter-diffusion technique and the three-dimensional structure of the EcPNP-7DHX complex was solved by molecular replacement at 2.51 Šresolution using an X-ray data set collected at the SPring-8 synchrotron-radiation facility, Japan. The crystals belonged to space group P6122, with unit-cell parameters a = b = 120.370, c = 238.971 Å, and contained three subunits of the hexameric enzyme molecule in the asymmetric unit. The 7DHX molecule was located with full occupancy in the active site of each of the three crystallographically independent enzyme subunits. The position of 7DHX overlapped with the positions occupied by purine bases in similar PNP complexes. However, the orientation of the 7DHX molecule differs from those of other bases: it is rotated by ∼180° relative to other bases. The peculiarities of the arrangement of 7DHX in the EcPNP active site are discussed.

A novel amperometric enzyme inhibition biosensor based on xanthine oxidase immobilised onto glassy carbon electrodes for bisphenol A determination.

A novel and simple biosensor for the determination of bisphenol A (BPA) based on xanthine oxidase (XOD) enzymatic inhibition has been developed. The biosensor was prepared from xanthine oxidase immobilised by crosslinking with glutaraldehyde, with hypoxanthine as enzyme substrate, and was successfully applied to the determination of BPA using fixed potential amperometry. Biosensor performance was optimised with respect to the applied potential, influence of pH of the electrolyte solution, XOD loading and the substrate concentration. The enzyme inhibition mechanism was evaluated from Cornish-Bowden plus Dixon plots and was found to be reversible and competitive with an apparent inhibition constant of 8.15 nM. Under optimised conditions, the determination of BPA can be achieved in the linear range up to 41 nM with a detection limit of 1.0 nM, which is equal to the lowest reported in the literature, with very good repeatability and reproducibility. The selectivity of the biosensor was evaluated by performing an interference study and found to be excellent; and stability was investigated. It was successfully applied to the detection of BPA in mineral water and in river water.

Performance comparison between multienzymes loaded single and dual electrodes for the simultaneous electrochemical detection of adenosine and metabolites in cancerous cells.

The analytical performance of the multi enzymes loaded single electrode sensor (SES) and dual electrode sensor (DES) was compared for the detection of adenosine and metabolites. The SES was fabricated by covalent binding of tri-enzymes, adenosine deaminase (ADA), purine nucleoside phosphorylase (PNP), and xanthine oxidase (XO) along with hydrazine (Hyd) onto a functionalized conducting polymer [2,2:5,2-terthiophene-3-(p-benzoic acid)] (pTTBA). The enzyme reaction electrode in DES was fabricated by covalent binding of ADA and PNP onto pTTBA coated on Au nanoparticles. The detection electrode in DES was constructed by covalent binding of XO and Hyd onto pTTBA coated on porous Au. Due to the higher amount (3.5 folds) of the immobilized enzymes and Hyd onto the DES than SES, and the lower Michaelis constant (Km) value for DES (28.7 µM) compared to SES (36.1 µM), the sensitivity was significantly enhanced for the DES (8.2 folds). The dynamic range obtained using DES was from 0.5 nM to 120.0 µM with a detection limit of 1.43 nM ±â€¯0.02, 0.76 nM ±â€¯0.02, and 0.48 nM ±â€¯0.01, for adenosine (AD), inosine (IN), and hypoxanthine (Hypo) respectively. Further, the DES was coupled with an electrochemical potential modulated microchannel for the separation and simultaneous detection of AD, IN, and Hypo in an extracellular matrix of cancerous (A549) and non-cancerous (Vero) cells. The sensor probe confirms a higher basal level of extracellular AD and its metabolites in cancer cells compared to normal cells. In addition, the effect of dipyridamole on released adenosine in A549 cells was investigated.