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This paper investigates the impact of taurine as an additive on the structural and functional stability of urate oxidase. First, the effect of the processing parameters for the stabilization of Urate Oxidase (UOX) using taurine was examined using the response surface methodology (RSM) and the central composite design (CCD) model. Also, the study examines thermodynamic and kinetic parameters as well as structural changes of urate oxidase with and without taurine. Fluorescence intensity changes indicated static quenching during taurine binding. The obtained result indicates that taurine has the ability to preserve the native structural conformation of UOX. Furthermore, molecular dynamics simulation is conducted in order to get insights into the alterations in the structure of urate oxidase in the absence and presence of taurine under optimal conditions. The molecular dynamics simulation section investigated the formation of hydrogen bonds (H-bonds) between different components as well as analysis of root mean square deviation (RMSD), root mean square fluctuations (RMSF) and secondary structure. Lower Cα-RMSD and RMSF values indicate greater stabilization of the taurine-treated UOX structure compared to the free enzyme. The results of molecular docking indicate that the binding of taurine to the UOX enzyme through hydrophobic interactions is associated with a negative value for the Gibbs free energy. [ABSTRACT FROM AUTHOR]

Purpose: Ureides, allantoin and allantoate, are N-rich compounds used for N transport in nodulated ureide legumes. Here, we investigated their role in response of Phaseolus vulgaris and Pisum sativum, representing ureide and amide legumes, respectively, to Cd toxicity. Methods: First, ureide content and ureide metabolism in P. vulgaris and P. sativum grown under control conditions and treated with 50 μM CdCl2 for 48 hours was investigated. Then, the effect of exogenous allantoin and its precursor, uric acid, on Cd-related oxidative lesion was examined in both legumes. Results: Cd increased the content of both ureides only in the leaves of P. vulgaris, which was consistent with transcript levels and activity of ureide metabolic enzymes, and was accompanied by an increase in uric acid content. In P. sativum leaves, Cd increased the activity of ureide biosynthesis enzymes and decreased the activity of ureide degradation enzymes, although the uric acid content did not change, while the allantoin and allantoate contents were significantly reduced. Exogenous uric acid and allantoin suppressed Cd-induced H2O2 and O2 accumulation and alleviated the effects of oxidative damage measured by RNA degradation, chlorophyll and malondialdehyde content in both legumes. Conclusions: P. sativum use allantoin and uric acid as antioxidant agents to mitigate Cd-related oxidative tissue damage. In P. vulgaris, the involvement of the ureide pathway in Cd-induced N salvage and recycling is rather a priority. [ABSTRACT FROM AUTHOR]

The study was to screen strains with high production of urate oxidase from duck feces and provide a new solution for alleviating and treating hyperuricemia in poultry. The strain that could produce transparent circle on uric acid screening medium was screened from fresh feces of healthy meat ducks. The enzyme production activity of the strain was determined by plate spot planting method, and the molecular identification was performed by 16S rDNA sequencing. The test strain mix was fed to Cherry Valley ducks to evaluate the feeding effect, and the effect of candidate strains on high uric acid Cherry Valley ducks was evaluated. The results showed that 56 strains producing uric acid oxidase were screened, among which one strain had the ability to produce protease and seven strains had the ability to produce amylase. 91% of the strains were gram negative, 27 of which were identified by molecular analysis, and 15 were of the Alcaligenes faecalis. Strains AS1 and P56 had the highest homology with Alcaligenes faecalis and Pseudomonas guariconensis, respectively. Strains AS1 and P56 had no significant effect on daily feed intake and body weight of ducks. AS1 could significantly reduce the serum uric acid concentration of hyperuricemic ducks (P<0.05). The experiment indicates that using the uric acid screening medium, the high-yield urate oxidase strain can be selected from the feces of healthy meat ducks, and AS1 can be used as a candidate strain to provide a reference for the subsequent mitigation and treatment of poultry hyperuricemia. [ABSTRACT FROM AUTHOR]

Introduction: SEL-212 is a developmental treatment for uncontrolled gout characterized by serum uric acid (sUA) levels ≥ 6 mg/dl despite treatment. It comprises a novel PEGylated uricase (SEL-037; also called pegadricase) co-administered with tolerogenic nanoparticles containing sirolimus (rapamycin) (SEL-110; also called ImmTOR®), which mitigates the formation of anti-drug antibodies (ADAs) against uricase and SEL-037 (PEGylated uricase), thereby enabling sustained sUA control (sUA < 6 mg/dl). The aim of this study was to identify appropriate dosing for SEL-037 and SEL-110 for use in phase 3 clinical trials. Methods: This open-label phase 2 study was conducted in adults with symptomatic gout and sUA ≥ 6 mg/dl. Participants received five monthly infusions of SEL-037 (0.2 or 0.4 mg/kg) alone or in combination with three or five monthly infusions of SEL-110 (0.05–0.15 mg/kg). Safety, tolerability, sUA, ADAs, and tophi were monitored for 6 months. Results: A total of 152 adults completed the study. SEL-037 alone resulted in rapid sUA reductions that were not sustained beyond 30 days in most participants due to ADA formation and loss of uricase activity. Levels of ADAs decreased with increasing doses of SEL-110 up to 0.1 mg/kg, with anti-uricase titers < 1080 correlating with sustained sUA control and reductions in tophi. Overall, 66% of evaluable participants achieved sUA control at week 20 following five monthly doses of SEL-037 0.2 mg/kg + SEL-110 0.1–0.15 mg/kg, whereas only 26% achieved sUA control at week 20 when SEL-110 was withdrawn after week 12. Compared to other dose combinations, SEL-037 0.2 mg/kg + SEL-110 0.15 mg/kg achieved the greatest sUA control at week 12 and was well-tolerated with no safety concerns. Conclusion: Results provide continued support for the use of multiple monthly administrations of SEL-037 0.2 mg/kg + SEL-110 0.1-0.15 mg/kg in clinical trials for SEL-212. Trial Registration: ClinicalTrials.gov identifier, NCT02959918. [ABSTRACT FROM AUTHOR]

Management of tumor lysis syndrome (TLS) associated with cancer chemotherapy for malignant tumors is important because of its potentially fatal course. The use of rasburicase, a recombinant urate oxidase, is recommended for TLS; however, because rasburicase is an enzymatic drug, one should be cautious of anaphylaxis during administration. Using claims data in Japan, we investigated the rate of rasburicase re-administration and the occurrence of anaphylaxis during re-administration in patients with hematopoietic malignancies in a multicenter setting. Re-administration of rasburicase was defined as administration after an interval of 21 days from the first dose. Of 373 patients, 18 were re-administered rasburicase (re-administration rate: 4.8%). No patient developed anaphylaxis. The median number of days from the first to the last dose of rasburicase was 256.5 days (interquartile range: 138.8–455.8 days). The median daily dose was 7.5 mg (4.5–11.3 mg), and the median total dose was 33.8 mg (19.1–64.1 mg). This claims database analysis revealed that the re-administration rate of rasburicase was low in Japanese patients with hematopoietic malignancies, suggesting that rasburicase was being used appropriately, and that associated anaphylaxis was not observed. [ABSTRACT FROM AUTHOR]

In a new approach, we considered the special affinity between Ni and poly-histidine tags of recombinant urate oxidase to utilize Ni-MOF for immobilizing the enzyme. In this study, a carbon paste electrode (CPE) was modified by histidine-tailed urate oxidase (H-UOX) and nickel-metal–organic framework (Ni-MOF) to construct H-UOX/Ni-MOF/CPE, which is a rapid, sensitive, and simple electrochemical biosensor for UA detection. The use of carboxy-terminal histidine-tailed urate oxidase in the construction of the electrode allows the urate oxidase enzyme to be positioned correctly in the electrode. This, in turn, enhances the efficiency of the biosensor. Characterization was carried out by X-ray diffraction (XRD), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), Brunauer–Emmett–Teller (BET), and field emission scanning electron microscopy (FE-SEM). At optimum conditions, the biosensor provided a short response time, linear response within 0.3–10 µM and 10–140 µM for UA with a detection limit of 0.084 µM, repeatability of 3.06%, and reproducibility of 4.9%. Furthermore, the biosensor revealed acceptable stability and selectivity of UA detection in the presence of the commonly coexisted ascorbic acid, dopamine, L-cysteine, urea, and glucose. The detection potential was at 0.4 V vs. Ag/AgCl. [ABSTRACT FROM AUTHOR]

Introduction: Gout, a common comorbidity of chronic kidney disease (CKD), is associated with high morbidity and healthcare utilization. However, a large proportion of gout remains undermanaged or untreated which may lead to worse patient outcomes and greater healthcare costs. This study estimates the present and future health and economic burden of controlled and uncontrolled gout in a virtual United States (US) CKD population.A validated microsimulation model was used to project the burden of gout in patients with CKD in the USA through 2035. Databases were utilized to build a virtual CKD population of “individuals” with controlled or uncontrolled gout. Modelling assumptions were made on the basis of the literature, which was sparse in some cases. Health and economic outcomes with the current care (baseline) scenario were evaluated, along with potential benefits of urate-lowering intervention scenarios.The prevalence of comorbid gout and CKD in the USA was projected to increase by 29%, from 7.9 million in 2023 to 9.6 million in 2035 in the baseline scenario. Gout flares, tophi, and comorbidity development were also projected to increase markedly through 2035, with the economic burden of gout in the CKD population subsequently increasing from $38.9 billion in 2023 to $47.3 billion in 2035. An increased use of oral urate-lowering therapies in undermanaged patients, and pegloticase use in patients refractory to oral urate-lowering therapies were also project to result in 744,000 and 353,000 fewer uncontrolled gout cases, respectively, by 2035. Marked reductions in complications and costs ensued.This study projected a substantial increase in comorbid gout and CKD. However, improved use of urate-lowering interventions could mitigate this growth and reduce the health and economic burdens of gout.Methods: Gout, a common comorbidity of chronic kidney disease (CKD), is associated with high morbidity and healthcare utilization. However, a large proportion of gout remains undermanaged or untreated which may lead to worse patient outcomes and greater healthcare costs. This study estimates the present and future health and economic burden of controlled and uncontrolled gout in a virtual United States (US) CKD population.A validated microsimulation model was used to project the burden of gout in patients with CKD in the USA through 2035. Databases were utilized to build a virtual CKD population of “individuals” with controlled or uncontrolled gout. Modelling assumptions were made on the basis of the literature, which was sparse in some cases. Health and economic outcomes with the current care (baseline) scenario were evaluated, along with potential benefits of urate-lowering intervention scenarios.The prevalence of comorbid gout and CKD in the USA was projected to increase by 29%, from 7.9 million in 2023 to 9.6 million in 2035 in the baseline scenario. Gout flares, tophi, and comorbidity development were also projected to increase markedly through 2035, with the economic burden of gout in the CKD population subsequently increasing from $38.9 billion in 2023 to $47.3 billion in 2035. An increased use of oral urate-lowering therapies in undermanaged patients, and pegloticase use in patients refractory to oral urate-lowering therapies were also project to result in 744,000 and 353,000 fewer uncontrolled gout cases, respectively, by 2035. Marked reductions in complications and costs ensued.This study projected a substantial increase in comorbid gout and CKD. However, improved use of urate-lowering interventions could mitigate this growth and reduce the health and economic burdens of gout.Results: Gout, a common comorbidity of chronic kidney disease (CKD), is associated with high morbidity and healthcare utilization. However, a large proportion of gout remains undermanaged or untreated which may lead to worse patient outcomes and greater healthcare costs. This study estimates the present and future health and economic burden of controlled and uncontrolled gout in a virtual United States (US) CKD population.A validated microsimulation model was used to project the burden of gout in patients with CKD in the USA through 2035. Databases were utilized to build a virtual CKD population of “individuals” with controlled or uncontrolled gout. Modelling assumptions were made on the basis of the literature, which was sparse in some cases. Health and economic outcomes with the current care (baseline) scenario were evaluated, along with potential benefits of urate-lowering intervention scenarios.The prevalence of comorbid gout and CKD in the USA was projected to increase by 29%, from 7.9 million in 2023 to 9.6 million in 2035 in the baseline scenario. Gout flares, tophi, and comorbidity development were also projected to increase markedly through 2035, with the economic burden of gout in the CKD population subsequently increasing from $38.9 billion in 2023 to $47.3 billion in 2035. An increased use of oral urate-lowering therapies in undermanaged patients, and pegloticase use in patients refractory to oral urate-lowering therapies were also project to result in 744,000 and 353,000 fewer uncontrolled gout cases, respectively, by 2035. Marked reductions in complications and costs ensued.This study projected a substantial increase in comorbid gout and CKD. However, improved use of urate-lowering interventions could mitigate this growth and reduce the health and economic burdens of gout.Conclusions: Gout, a common comorbidity of chronic kidney disease (CKD), is associated with high morbidity and healthcare utilization. However, a large proportion of gout remains undermanaged or untreated which may lead to worse patient outcomes and greater healthcare costs. This study estimates the present and future health and economic burden of controlled and uncontrolled gout in a virtual United States (US) CKD population.A validated microsimulation model was used to project the burden of gout in patients with CKD in the USA through 2035. Databases were utilized to build a virtual CKD population of “individuals” with controlled or uncontrolled gout. Modelling assumptions were made on the basis of the literature, which was sparse in some cases. Health and economic outcomes with the current care (baseline) scenario were evaluated, along with potential benefits of urate-lowering intervention scenarios.The prevalence of comorbid gout and CKD in the USA was projected to increase by 29%, from 7.9 million in 2023 to 9.6 million in 2035 in the baseline scenario. Gout flares, tophi, and comorbidity development were also projected to increase markedly through 2035, with the economic burden of gout in the CKD population subsequently increasing from $38.9 billion in 2023 to $47.3 billion in 2035. An increased use of oral urate-lowering therapies in undermanaged patients, and pegloticase use in patients refractory to oral urate-lowering therapies were also project to result in 744,000 and 353,000 fewer uncontrolled gout cases, respectively, by 2035. Marked reductions in complications and costs ensued.This study projected a substantial increase in comorbid gout and CKD. However, improved use of urate-lowering interventions could mitigate this growth and reduce the health and economic burdens of gout. [ABSTRACT FROM AUTHOR]

The stability of the tetrameric enzyme urate oxidase in complex with excess of 8‐azaxanthine was investigated either under high hydrostatic pressure per se or under a high pressure of argon. The active site is located at the interface of two subunits, and the catalytic activity is directly related to the integrity of the tetramer. This study demonstrates that applying pressure to a protein–ligand complex drives the thermodynamic equilibrium towards ligand saturation of the complex, revealing a new binding site. A transient dimeric intermediate that occurs during the pressure‐induced dissociation process was characterized under argon pressure and excited substates of the enzyme that occur during the catalytic cycle can be trapped by pressure. Comparison of the different structures under pressure infers an allosteric role of the internal hydrophobic cavity in which argon is bound, since this cavity provides the necessary flexibility for the active site to function. [ABSTRACT FROM AUTHOR]

Background and Objective: Streptomyces are gram-positive and aerobic bacterial strains that are isolated from different sources. Streptomyces have the ability to produce secondary metabolites and biologically active substances and are therefore very important in the field of biocontrol. Urate oxidase is a microbial enzyme product that can be extracted from a variety of sources, including streptomycin. In the present study, cloning of the urate oxidase gene isolated from seawater streptomycosis was performed in Escherichia coli Origami bacteria. Methods: In this descriptive study, a total of 60 water and sediment samples were collected from different depths of the Caspian Sea coast in Mazandaran province, Iran. The Geram, staining methyl red, VP, citrate, starch hydrolysis, casein hydrolysis, nitrate reduction, oxidase and catalase tests were performed to identify and isolate Streptomyces. The urate oxidase gene was cloned using the T-A cloning method using the PTG-19 vector inside the host of Escherichia coli Origami. The expression of cloned genes in recombinant colonies was investigated by Real-Time PCR. The phylogenetic tree was drawn using clustalX and Mega5 software. Results: Screening of marine water samples identified 12 isolated streptomyces, all of which had the urate oxidase gene. The expression of urate oxidase gene in Escherichia coli Origami was confirmed by Real-Time PCR. The results of phylogenetic studies identified some close relatives of Streptomyces as candidates for subsequent studies. Conclusion: Streptococcus bacteria can be considered as a rich source of secondary metabolites with many applications and can be used as a native to produce the enzyme urate oxidase. By using different cloning hosts and examining optimal production conditions, this strain can be a candidate for future studies to develop antimicrobial drugs and compounds. [ABSTRACT FROM AUTHOR]

This study presents an electrochemical biosensor developed for uric acid (UA) determination using carbon paste electrode (CPE) modified with copper (II) oxide (CuO) particles and urate oxidase (UOx) enzyme. Base CPE is prepared using a multi-walled carbon nanotube (MWCNT) and a polydimethylsiloxane (PDMS) binder. The main sensing process is based on the oxidation of UA into 5-hydroxyisourate (HIU) as catalyzed by UOx, forming H2O2 as byproduct, and then the H2O2 then the H2O2 reduction-oxidation (redox) reaction converts CuO to form Cu2O; the amount of H2O2 and hence UA in the sample is measured by the oxidative current measured for the conversion of Cu2O back to CuO. Cyclic Voltammetry (CV) measurements revealed that the activity of UOx was retained with an apparent Michaelis constant (Kmapp) to be equal to 41.46 µM. Differential Pulse Voltammetry (DPV) measurements of UA using UOx-CuO-CPE showed a linear response ranging from 10 µM to 79.4 µM UA with a limit of detection (LOD) determined to be equal to 8.82 µM. UOx-CuO-CPE was shown to be selective towards UA even in the presence of creatinine, xanthine, and glucose. Furthermore, UOx-CuO-CPE was shown to be reusable (3.28% RSD), and its fabrication is repeatable using single factor Analysis of Variance (ANOVA) [F(1.396) < Fcritical(5.143)]. UOx-CuO-CPE was also shown to be stable even after five weeks of storage using the two-sample t-test [t(0.156) < tcritical(4.303)]. Based on a recovery test using synthetic urine sample, this study showed the applicability of UOx-CuO-CPE in the detection of UA in human urine with 90.27%-102.03% recovery (n = 3). [ABSTRACT FROM AUTHOR]

A high serum uric acid (SUA) concentration is associated with hyperuricemia (HUA) and gout. In order to obtain long-acting therapeutic effect, correction of purine metabolism at genetic level is advantageous. For this purpose, we expressed three "human-like" urate oxidases in human hepatocytes (HL-7702) by lentivirus-mediated transduction. Enzymatic assay revealed that the recombinant urate oxidases expressed in HL-7702 cells were functionally active. Electron microscopy study showed that the recombinant enzymes were localized to peroxisome and formed distinct crystalloid core structures as in other mammal cells. Although similar rate of uric acid degradation was observed for all recombinant urate oxidases, HL-7702-pLVX-UOX 83 cells and HL-7702-pLVX-UOX 214/217 cells retained more cell viability compared with HL-7702-pLVX-UOX PBC at high uric acid level. This study provides a new direction for the treatment of gout and hyperuricemia. [ABSTRACT FROM AUTHOR]

• Intracellular production of recombinant A. flavus Urate Oxidase (rUOX). • The downstream process (developed for high purity) provides the highest known yield. • Response surface methodology was employed to optimize the polishing step. • Different characterizations were conducted to determine final product quality. Recombinant A. flavus Urate Oxidase (rUOX), which catalyzes uric acid to allantoin, is highly effective in treating tumor lysis syndrome (TLS). However, its production process is quite complicated, and its use as a therapy is often prohibitive because of the high cost. In this work, we demonstrate, for the first time, a new bioprocess for the production of rUOX expressed in P. pastoris GS115 intracellularly in a soluble and active form, which aims to develop biosimilars that address the current limitations. For the downstream process, the homogenate clarification and intermediate purification were rationally designed with regards to protein characteristics, including polyethyleneimine precipitation, ammonium sulfate precipitation, and flow-through module anion-exchange chromatography. Response surface methodology (RSM) was conducted to optimize the cation exchanger polishing step to determine the appropriate conditions that satisfy the purity and yield requirements. Finally, 2.2 g rUOX (99% purity) was obtained from the 1 l fermentation broth. Various characterizations were carried out to confirm rUOX's similarity to its commercial counterpart with regards to both physicochemical and enzymatic aspects. Overall, the rUOX derived from P. pastoris has the potential to be used in therapeutic applications. [ABSTRACT FROM AUTHOR]

Twenty-five microbial isolates were investigated for uricase production on uric acid medium. All isolates were obtained from Jeddah, Saudi Arabia. The highest uricase producer was identified as Bacillus cereus SKIII. Using glucose peptone broth at pH 7.5, incubation temperature 30 °C for 3 days with shaking of 150 rpm were the best conditions for maximum enzyme production. Glucose and peptone were the best carbon and nitrogen sources. The molecular weight of the purified enzyme was34.5 KDa, and isoelectric point was 7.9. The optimum pH and temperature were pH 8.0 and 35 °C, respectively. It was stable at 35 °C for 60 min, but thermally inactivated at 60 °C after 60 min Its enzymatic activity was enhanced by Mg2+, Ca2+,Fe2+, Mn2+, Zn2+ions and inhibited by Co2+, Na+, Hg2+, Ag+ ions and EDTA at 1 mM. Uricase production was enhanced using UV mutation and the obtained mutant produced six times higher than the original isolate. An amplicon 900 bp of uricase gene (Pucl) was sequenced (accession number MF417635). No remarkable difference was noticed in B. cereus SKIII and SKm mutant nucleotide sequences. In conclusion, SKIII and SKm are promising strains in uricase production for biotechnological applications. [ABSTRACT FROM AUTHOR]

Present study focuses on determination of complex dielectric constant of biomolecules as function of frequency by means of surface plasmon resonance (SPR) technique without losing their biofunctionality. Surface plasmon modes have been excited in Kretschmann configuration at interface of ZnO-Au thin films. Various biomolecules (glucose oxidase, cholesterol oxidase, urease, and uricase) have been immobilized successfully on surface of ZnO thin film by electrostatic interaction. SPR reflectance curves for all biomolecules were recorded separately at different wavelengths (407-635 nm). Complex dielectric constant was determined by fitting the experimental SPR data with Fresnel's equations. Dielectric constant of all biomolecules shows frequency dispersion and attributed to ionic polarization. [ABSTRACT FROM AUTHOR]

This study presents an electrochemical biosensor developed for uric acid (UA) determination using carbon paste electrode (CPE) modified with copper (II) oxide (CuO) particles and urate oxidase (UOx) enzyme. Base CPE is prepared using a multi-walled carbon nanotube (MWCNT) and a polydimethylsiloxane (PDMS) binder. The main sensing process is based on the oxidation of UA into 5-hydroxyisourate (HIU) as catalyzed by UOx, forming H2O2 as byproduct, and then the H2O2 reduction-oxidation (redox) reaction converts CuO to form Cu2O; the amount of H2O2 and hence UA in the sample is measured by the oxidative current measured for the conversion of Cu2O back to CuO. Cyclic Voltammetry (CV) measurements revealed that the activity of UOx was retained with an apparent Michaelis constant (Km app) to be equal to 41.46 µM. Differential Pulse Voltammetry (DPV) measurements of UA using UOx-CuO-CPE showed a linear response ranging from 10 µM to 79.4 µM UA with a limit of detection (LOD) determined to be equal to 8.82 µM. UOx-CuO-CPE was shown to be selective towards UA even in the presence of creatinine, xanthine, and glucose. Furthermore, UOx-CuO-CPE was shown to be reusable (3.28% RSD), and its fabrication is repeatable using single factor Analysis of Variance (ANOVA) [F(1.396) < Fcritical(5.143)]. UOx-CuO-CPE was also shown to be stable even after five weeks of storage using the two-sample t-test [t(0.156) < tcritical(4.303)]. Based on a recovery test using synthetic urine sample, this study showed the applicability of UOx-CuO-CPE in the detection of UA in human urine with 90.27%-102.03% recovery (n = 3). [ABSTRACT FROM AUTHOR]

Therapeutic proteins are indispensable for treatment of various human diseases. However, intrinsic short serum half-lives of proteins are still big hurdles for developing new therapeutic proteins or expanding applications of existing ones. Urate oxidase (Uox) is a therapeutic protein clinically used for treatment of hyperuricemia. Due to its short half-life, its application for gout treatment requires prolonging the half-life in vivo. Conjugation of a fatty acid (FA), a serum albumin (SA) ligand, to therapeutic proteins/peptides is an emerging strategy to prolong serum half-life presumably via neonatal Fc receptor (FcRn)–mediated recycling. FA conjugation was proven effective for peptides and small proteins (less than 28 kDa), but not for Uox (140 kDa). We hypothesized that the intramolecular distance in the conjugate of FA and Uox is a critical factor for effective FcRn-mediated recycling. In order to control the intramolecular distance in the conjugate, we varied linker lengths between Uox and palmitic acid (PA). There was a linear correlation between the linker length and serum half-life of PA–conjugated Uox (Uox-PA) conjugates. The longer linker led to about 7-fold greater extension of serum half-life of Uox in mice than the unmodified Uox. The trend in serum half-life extension matched well with that in the tertiary structure formation of FcRn/SA/Uox-PA in vitro. These results demonstrate that the intramolecular distance in the conjugate of Uox and FA governs the stable formation of FcRn/SA/FA-conjugated protein and serum half-life extension in vivo. These findings would also contribute to development of effective FAconjugated therapeutic proteins. [ABSTRACT FROM AUTHOR]

Previous studies have demonstrated the effects of hyperuricemia on the damage to target organs, including the kidneys, joints and the heart. However, it is unclear whether hyperuricemia results in damage to the intestines. The aim of the present study was to investigate intestinal barrier dysfunction in a mouse model of hyperuricemia constructed by knocking out the urate oxidase (Uox) gene. The morphology of the intestine was assessed via hematoxylin and eosin, and alcian blue staining. The serum and intestinal tissue levels of uric acid, tumor necrosis factor (TNF)-α and interleukin (IL)-6, in addition to the presence of uremic toxins in the serum, were assessed. The levels of diamine oxidase (DAO), D-lactate (D-LAC) and endotoxins in the serum, which are markers of the intestinal permeability, were measured using ELISA. The expression of the intestinal tight junction proteins zona occludens-1 (ZO-1) and occludin were detected by reverse transcription-quantitative polymerase chain reaction, western blotting and immunohistochemical analysis. The Uox-knockout mice spontaneously developed hyperuricemia. Histopathological analysis indicated notable intestinal defects including sparse villi, mucosal edema and a declining mucus layer in hyperuricemic mice. The expression levels of ZO-1 and occludin in the intestines were downregulated, and the serum levels of DAO, D-LAC and endotoxins were higher in the hyperuricemic mice, compared with control mice. The serum and intestinal tissue levels of IL-6 and TNF-α were significantly increased. Additionally, the expression levels of the serum uremic toxins, serum creatinine, blood urea nitrogen were significantly increased in hyperuricemic mice compared with the control mice, while only a marked increase in indoxyl sulfate (IS) and p-cresol sulfate was reported. Collectively, the results of the present study suggested that intestinal barrier dysfunction and subsequent enhanced intestinal permeability may occur as a result of hyperuricemia in mice. Furthermore, we proposed that the loss of intestinal epithelium barrier function may be associated with uric acid-induced inflammatory responses; however, further investigation is required. [ABSTRACT FROM AUTHOR]

• A novel alkaline urate oxidase from Arxula adeninivorans NBRC 10858 was identified. • The enzyme showed its maximal activity at 40 °C and pH 10. • The enzyme showed higher turnover number and catalytic efficiency than existing ones. • It efficiently reduced the uric acid and purine content in beer, beef and yeast extract. • His 6 -tag fusion and G300D substitution may underlie its superior catalytic performance. This study reported a novel highly active alkaline urate oxidase (UOX) and demonstrated its application in reducing uric acid content of food under alkaline conditions. The UOX gene was cloned from Arxula adeninivorans NBRC 10858, and its N-terminally his 6 -tagged form (rUOX) was overexpressed in Escherichia coli. The rUOX displayed maximal activity at 40 °C and pH 10, kept more than 90% initial activity under alkaline conditions (pH 9–11) and more than 80% at temperatures below 55 °C. The apparent K m , turnover number (k cat) and catalytic efficiency (k cat / K m) values for the substrate uric acid were respective 29.15 µM, 151.16 s−1 and 5.19 s−1. μM−1, which are improvements over previously reported UOXs. The rUOX efficiently reduced uric acid and purine contents in beer, beef and yeast extract at pH 10, indicating a promising application in food with low purine and uric acid contents to prevent hyperuricemia and gout. [ABSTRACT FROM AUTHOR]

Hyperuricemia is a foundation of multiple diseases，and animal models are the basis for scientists to study new strategies against the disease, develop and evaluate novel drugs. The present article summarized the animal models which were used to study hyperuricemia, and grouped them into two classes, namely, models caused bydrugs and those by gene knock-out. Drugs used to induce hyperuricemia are purines like adenine, uricase inhibitors like potassium oxonate, and urate secretion inhibitors like ethambutol. Gene knock- out models established were targeted at Uoxor ABCG2 gene. In order toavoid disturbance fromdrugs, Uoxd eficient mice or rats are favorite model animals in future. [ABSTRACT FROM AUTHOR]

Hyperuricemia is a foundation of multiple diseases， and lowering serum uric acid is able to effectively prevent and treat the diseases such as gout， renal dysfunction and cardiovascular diseases. Recently， new achievements in study on hyperuricemia treatment and prevention were emerged，which needs to be summed up. At first， the present article briefly reviewed the synthesis and elimination of uric acid，and grouped the ways of prevention and treatment into three strategies: inhibiting synthesis， enhancing excretion， and promoting degradation. In the end， the article put forward that future strategy should focus on intestinal tract to lower serum uric acid by local rather than systematic administration. [ABSTRACT FROM AUTHOR]

Abstract Quaternary chalcopyrite, i.e., Cu 2 ZnSnS 4 (CZTS) nanoparticles films have been proposed as a novel matrix system for enzyme-based electrochemical biosensors providing a non-toxic, low-cost alternative for the fabrication of bioelectrodes. The easy tuneability of the band gap of CZTS by varying the cation ratio and size of nanoparticles facilitate to impart desirable electrical properties in the material. Butylamine capped spherical CZTS nanoparticles of size 15–16 nm and band gap 2.65 eV have been synthesized by colloidal hot injection technique. The films of CZTS onto ITO substrates are deposited using dip coating technique, and uricase enzyme have been immobilized onto CZTS films using EDC-NHS binding chemistry. Electrochemical analyses of this bioelectrode revealed that the uricase/CZTS/ITO/glass electrode exhibits good linearity over a wide range of 0–700 μM uric acid concentration with a limit of detection (LOD) of 0.066 µM. The low value of 0.13 × 10−4 M of Michaelis–Menten constant (K m) indicate the enhanced affinity of immobilized enzyme (uricase) towards uric acid. Thus, the present report confirms the promising application of the p-type CZTS thin film matrix for the realization of an electrochemical biosensor. Highlights • Synthesis of wide band gap CZTS using Butylamine capping ligand for bio-sensing. • Fabricating enzyme based, electrochemical sensing device for uric acid detection. • CZTS nanoparticles based thin film is used as a novel matrix system. • Amine coupling using EDC:NHS is explored for improved sensing capability. [ABSTRACT FROM AUTHOR]

Highlights • Plasma from Acid Citric Dextrose (ACD) tubes decreases measured Uric acid concentrations. • Citric acid in ACD tubes interferes with the uricase-based method of detection. • Scavenging of hydrogen peroxide by citric acid competes for the leuco dye substrate. • Clinicians should avoid tubes containing citric acid when measuring uric acid. Abstract Background Circulating uric acid (UA) is an important biomarker, not only in the detection and management of gout, but also in assessing the risk of related comorbidity. The impact of collection methods on clinical UA measurements has been the subject of limited study. After observing significant differences between UA concentrations of blood samples obtained by different collection tubes, we began examining the effects of exogenous tube components on measured UA concentrations. We aimed to: (1) demonstrate the variability in uricase-based UA measurements attributable to different collection methods and (2) identify factors influencing this variability. Methods Blood samples from human subjects were collected using Serum Separator Tubes (SST tubes), Acid Citrate Dextrose (ACD) tubes, and Sodium Citrate (SC) tubes. Circulating UA concentrations were measured by chemistry analyzers utilizing the uricase method. Absorbance assays were run in order to determine the effects of citric acid, sodium citrate, and dextrose on measured absorbance in the presence of leuco crystal violet dye, hydrogen peroxide, and peroxidase. Statistical analyses—including Student's T tests and ANOVA—were used to compare results. Results UA concentrations of blood samples collected in ACD tubes were significantly lower than those collected in SST tubes (P < 0.01). Samples collected in SC tubes trended towards lower UA measurements than samples collected in SST tubes, although this difference did not reach statistical significance (P = 0.06). Blood samples spiked with separate concentrations of sodium citrate (3.2 and 22.0 g/L), citric acid (8.0 g/L), and dextrose (24.5 g/L) demonstrated significantly lower UA measurements compared to controls (P < 0.01). Absorbance assays demonstrated that increasing concentrations of citric acid and sodium citrate—in the presence of leuco crystal violet, hydrogen peroxide, and peroxidase—decreased the amount of oxidized dye in the uricase method of UA measurement in a dose-dependent manner (P < 0.01). In contrast, dextrose did not significantly alter the amount of oxidized dye available. Discussion Our results indicate that citric acid obstructs accurate uricase-based UA measurement, providing falsely low values. Citric acid, a known antioxidant, scavenges hydrogen peroxide, a key intermediate using the uricase method. By scavenging hydrogen peroxide, citric acid decreases the amount of oxidized leuco dye leading to falsely low UA measurements. Therefore, collection tubes, like ACD and SC tubes, which contain concentrations of citric acid or its conjugate base sodium citrate should not be used to measure circulating UA levels when utilizing uricase-based measurement methods. [ABSTRACT FROM AUTHOR]

A simple, rapid, and effective colorimetric assay for the detection of uric acid (UA) has been built, using the MoS2 nanoflakes-catalyzed 3,3′,5,5′-tetramethylbenzidine (TMB)-H2O2 system. In the presence of oxygen and uricase, uric acid was oxidized specifically to produce H2O2. MoS2 nanoflakes synthesized by hydrothermal reaction could catalyze the oxidation of TMB by H2O2, and engendering the colorimetric signal. Finally, the change in the color from colorless to blue was seen with naked eye, indicating the presence or absence of UA. Under the optimized conditions, a linear relationship between the absorbance and UA concentration in the range of 0.5-100 μM (R2 = 0.996) with the limit of detection for 0.3 μM (S/N = 3). The proposed assay was successfully applied to the detection of UA in human serum with the recoveries over 94.54%. Thus, these results suggest that the UA assay-based MoS2-catalyzed TMB-H2O2 has great foreground for fast clinical diagnosis of gout without the need for advanced and costly equipment.ᅟ [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • Statistically designed L18 orthogonal array improved Bc-uricase production by 11.4%. • Validation and scale up in bioreactor under automated controlled conditions. • The purified clinical Bc-uricase may be applied for the treatment of gout disease. • The homology modelled structure of Bc -uricase displayed the conserved T-fold structure. Abstract The uricase production from Bacillus cereus after statistical optimization of process variables designed by L18 orthogonal array gave enhanced enzyme activity by 13% (29.7 U/mL) as compared with un-optimized medium (26.3 U/mL). The optimized process variables used for scale up of uricase production in a bioreactor gave 39.7 U/mL of uricase activity resulting 51% increase as compared with unoptimized medium. The Luedeking-Piret model classified the uricase production from Bacillus cereus as mixed-growth associated. The culture supernatant having uricase activity (7.7 U/mg) on purification by (NH 4) 2 SO 4 precipitation followed by anion-exchange and size-exclusion chromatography resulted 87 U/mg giving 11-fold purification. The purified uricase can be used for treating gout disease. The de novo amino acid sequence analysis of purified uricase by MALDI-TOF MS showed 301 amino acids and molecular mass of 34.4 kDa, which was corroborated by 35 kDa from SDS-PAGE. The phylogenetic tree generated by homologous amino acid sequences displayed the clustering of Bacillus uricase with fungal uricase. The secondary structure analysis of uricase by circular dichroism showed 18.6% α-helices and 27.4% β-strands. These results were corroborated by secondary structure prediction analysis by PsiPred. The homology modelling of uricase displayed the conserved T-fold structure. [ABSTRACT FROM AUTHOR]

In clinical diagnosis, monitoring of uric acid (UA) is generally realized by combining uricase with natural peroxidase. The use of bio-enzymes, however, shadows some highlights of these methods due to their vulnerable activities against environments. Herein, we report a novel biosensor for the natural enzyme-free colorimetric detection of UA by using CoP nanosheet arrays grown on Ni foam (NF) as a monolithic peroxidase mimic. The integrated nanozyme can be put into and taken out from reaction systems conveniently with only tweezers, making it possible for on-demand analysis. As demonstrated, the obtained CoP/NF exhibits outstanding peroxidase-like activity to trigger the oxidation reaction of colorless 3,3′5,5′-tetramethylbenzidine (TMB) to a blue product (TMBox) mediated by H 2 O 2 . It is found that the blue TMBox can be reduced to colorless TMB again by UA selectively, thus the presence of UA in solutions will suppress the color reaction of TMB. Based on this principle, an uricase-free biosensor is developed for the photometric determination of UA, providing a wide detection range of 1–200 μM and a limit of detection down to 1.0 μM. In addition, the fabricated biosensor can be applied for measuring UA in clinical samples with merits of simple operation and good reliability, exhibiting its great promise in clinical diagnosis. [ABSTRACT FROM AUTHOR]

Objective: Observational studies have shown that increased plasma urate is associated with lower risk of Parkinson's disease (PD), but these studies were not designed to test causality. If a causal relationship exists, then modulating plasma urate levels could be a potential preventive avenue for PD. We used a large two-sample Mendelian randomization (MR) design to assess for a causal relationship between plasma urate and PD risk.Methods: We used a genetic instrument consisting of 31 independent loci for plasma urate on a case-control genome-wide association study data set, which included 13,708 PD cases and 95,282 controls. Individual effect estimates for each SNP were combined using the inverse-variance weighted (IVW) method. Two additional methods, MR-Egger and a penalized weighted median (PWM)-based approach, were used to assess potential bias attributed to pleiotropy or invalid instruments.Results: We found no evidence for a causal relationship between urate and PD, with an effect estimate from the IVW method of odds ratio (OR) 1.03 (95% confidence interval [CI], 0.88-1.20) per 1-standard-deviation increase in plasma urate levels. MR Egger and PWM analyses yielded similar estimates (OR, 0.99 [95% CI, 0.83-1.17] and 0.99 [95% CI, 0.86-1.14], respectively).Interpretation: We did not find evidence for a linear causal protective effect by urate on PD risk. The associations observed in previous observational studies may be, in part, attributed to confounding or reverse causality. In the context of the present findings, strategies to elevate circulating urate levels may not reduce overall PD risk. Ann Neurol 2018;84:191-199. [ABSTRACT FROM AUTHOR]

Objective: Urate is a potent antioxidant, and high plasma urate has been associated with lower incidence of Parkinson's disease (PD) in epidemiological studies. We tested the hypothesis that high concentrations of plasma urate are associated with low incidence of PD.Methods: We performed observational and genetic analyses using plasma urate and the urate SLC2A9 rs7442295 and ABCG2 rs2231142 genotype in >102,000 individuals from the CGPS (Copenhagen General Population Study). Information on PD and mortality was from national patient and death registries. Incidences of PD were calculated using Cox regression, Fine and Gray competing-risks regression, and instrumental variable analyses.Results: In total, 398 individuals were diagnosed with PD, of which 285 were incident cases. The multivariable adjusted hazard ratio for PD was 0.56 (95% confidence interval [CI], 0.41-0.77) for the highest versus the lowest tertile of plasma urate (p for trend across 3 groups, 8 × 10-5 ). Each one-allele increase in the combined allele score was associated with 19μmol/l (95% CI, 18.5-19.9) higher plasma urate. In observational analyses, a 50μmol/l higher plasma urate was associated with a hazard ratio of 0.85 (0.77-0.92) for PD; in instrumental variable analyses, 50μmol/l higher plasma urate was associated with an odds ratio of 1.20 (0.85-1.71) for PD.Interpretation: High plasma urate was associated with lower risk of PD in observational analyses; however, in instrumental variable analysis, high plasma urate was not associated with low risk of PD. Thus, our data do not support a causal relationship between high plasma urate and low risk of PD. Ann Neurol 2018;84:178-190. [ABSTRACT FROM AUTHOR]

Microbes can be used to improve the quality of fermented beverages and food. Colletotrichum camelliae is one of the most dominant fungi associated with tea leaves. In the present study, black tea produced from the autumn leaves of Yellowish Yinghong 9 was treated with C. camelliae urate oxidase (CcUOX) to determine whether this treatment could improve tea quality. The effects of recombinant CcUOX on several black tea quality parameters were assessed, such as liquor color, aroma, and levels of theabrownins, thearubigins, total polyphenols, catechins, caffeine, xanthine, and hypoxanthine. CcUOX treatment significantly affected the aroma composition and volatile compound profiles. Specifically, volatiles in the benzenoid/phenylpropanoid, fatty acid metabolism, and amino acid pathways were increased in CcUOX-treated black tea, whereas levels of volatiles in the terpineol and carotenoid pathways were reduced. This suggested that the balance of aroma constituents in Yellowish Yinghong 9 black tea was affected by treatment with CcUOX. The present study provides evidence that pre-treatment of black tea with a single enzyme from a common tea-associated microbe could significantly improve autumn tea quality, validating this method for use in further studies of tea improvement. • Treatment with a fungal-derived u rate oxidase (UOX) could improve black tea quality. • CcUOX treatment enhanced red brilliance, red evenness, sweet and mellow flavor, and high and sweet flavor of tea liquor. • CcUOX treatment significantly increased theabrownins, caffeine, xanthine, EGCG, and CG abundance. • CcUOX significantly increased concentrations of volatiles in the phenylpropanoid and amino acid pathways. [ABSTRACT FROM AUTHOR]

Low energy ultrasound irradiation was used to enhance co-production of enzymes uricase and alkaline protease using Bacillus licheniformis NRRL 14209. Production of uricase and alkaline protease was evaluated for different ultrasound parameters such as ultrasound power, time of irradiation, duty cycle and growth stage of organisms at which irradiation is carried out. Maximum uricase production of 0.825 U/mL and alkaline protease of 0.646 U/mL have been obtained when fermentation broth was irradiated at 6 h of growth stage with 60 W power for 15 min of duration having 40% of duty cycle. The enzyme yield was found to be enhanced by a factor of 1.9–3.8 and 1.2–2.2 for uricase and alkaline protease respectively. Nevertheless, intracellular uricase was also observed in a fermentation broth after ultrasonic process intensification. The results indicate the effectiveness of low frequency ultrasound in improving enzyme yields with a vision of commercial applicability of the process. [ABSTRACT FROM AUTHOR]

Abstract: A feeding trial was conducted with juvenile rainbow trout (15–16 g initial weight) to assess the effects of including single‐cell protein (SCP) produced from Methylobacterium extorquens in trout feeds. Three isonitrogenous and isoenergetic diets were produced: a control diet and two experimental diets containing 5% or 10% bacterial protein meal replacing soybean meal. Triplicate tanks, each containing 35 fish, were fed each diet to apparent satiation in a constant‐temperature (15°C), flow‐through tank system for 12 weeks. No statistically significant differences in final fish weight or other fish growth parameters were observed. Similarly, feed efficiency parameters showed no significant differences among groups. Nutrient retention indices (protein, fat, energy) were relatively high and similar among fish in each dietary treatment group, as were whole body proximate compositions. Fish survival was high, with a small but statistically significant increase for the 10% SCP diet. Overall, results demonstrate that SCP from M. extorquens is a safe and effective alternative protein for rainbow trout diets at the low inclusion levels tested. Slightly lower weight gain in fish fed the 10% SCP diet was largely due to lower feed intake, suggesting that adding palatability‐enhancing ingredients to feeds may allow higher levels of M. extorquens SCP to be used without compromising fish growth. [ABSTRACT FROM AUTHOR]

Transient leukemoid reactions are well-documented in the neonatal intensive care unit, however, hyperleukocytosis greater than 100 x 109/L remains a rare entity in premature neonates. We report the development of extreme hyperleukocytosis in an extremely low birth weight premature neonate born at 24-2/7 weeks of gestational age due to premature rupture of membranes in a mother who had received antenatal corticosteroids. The patient subsequently developed gross dyselectrolytemia similar to tumor lysis syndrome. Infectious workup remained unrevealing, flow cytometry of the peripheral blood showed no immunophenotypic evidence of acute leukemia, and a normal female XX karyotype was confirmed. A single dose of rasburicase was administered, with rapid and safe resolution of hyperuricemia, allowing for maintenance of kidney function. The patient was treated for presumed sepsis, although cultures remained negative. The white blood cell count gradually normalized over the first two weeks of life. The patient required treatment for a patent ductus arteriosus and was severely ventilator dependent for 33 days post-partum. To our knowledge, this is the first documented case wherein electrolyte abnormalities similar to those seen in tumor lysis syndrome had complicated the course of a leukemoid reaction in an extremely premature neonate necessitating the use of rasburicase. [ABSTRACT FROM AUTHOR]

Aims Expression and purification of recombinant Urate oxidase as well as other pharmaceutical proteins are a costly process. The use of non-chromatographic methods in the purification process can reduce production costs. So, the aim of this study was cloning, expression, and purification of recombinant urate oxidase, using intein sequence and elastin-like protein (ELP). Materials & Methods In the present experimental research, the synthetic urate oxidase gene was cloned in downstream of ELP–Intein in an expression plasmid. After cultivating bacteria containing urate oxidase expression plasmid, the soluble fraction of cell lysate was separated from insoluble portion, using centrifugation. The recombinant urate oxidase was precipitated by adding salt and incubation at 37°C. The resulting precipitant was resuspended in cleavage buffer and the urate oxidase was separated from ELP through Intein moiety. Adding salt for the second time and incubation at 37°C resulted in separation of urate oxidase from fusion partner. After expressing and purifying the urate oxidase, purity and biological activity of recombinant urate oxidase were compared to standard drug. Findings The recombinant urate oxidase was expressed and purified using ELP and Intein tags as fusions partners. The rescombinant enzyme showed a purity of %90 equal to 1.89 mg.ml-1 based on protein concentration and 1.64 mg.ml-1 based on activity. Conclusion Using Intein sequence and Elastin-like protein, together with the Urate Oxidase enzyme helps to express and purify the enzyme without need for a chromatographic method. [ABSTRACT FROM AUTHOR]

Allantoin, the final product of urate degradation in non-hominoid mammals, forms in vivo through an enzyme-dependent pathway whose restoration has been proposed for the treatment of hyperuricemic conditions. Non-enzymatic urate oxidation to allantoin also occurs, making it a potential biomarker of oxidative stress in human physiology and pathology. This justifies the quest for fast and accurate methods for allantoin quantification overcoming the limitations of the current approaches, ranging from chromatographic to mass spectrometry techniques. We propose a simple fluorimetric assay that includes an enzymatic step for the conversion of S -allantoin to allantoate, followed by acid hydrolysis of the latter into glyoxylate in the presence of resorcinol. The lactone forming upon reaction of glyoxylate with resorcinol, in basic conditions, exhibits strong and stable fluorescence emission in the visible range. The assay proved to be specific for allantoin, with a linear response in the range of physiological values for several biological fluids, such as serum, urine, and saliva. We also verified the lack of reactivity by the most common interferents, including urate and its degradation intermediates. The assay, which does not require sophisticated instrumentation other than a conventional spectrofluorometer or a lab bench plate reader, was implemented on a solid support, exploiting enzyme encapsulation in a silica gel matrix, to allow reusability and improve stability over time. [ABSTRACT FROM AUTHOR]

This work investigates the use of electrochemical platforms containing polymeric films derived from 4-aminosalicylic acid (4-ASA) modified with the enzyme urate oxidase (UOx) for quantification of uric acid (UA) in urine samples. Electropolymerization of 4-ASA was investigated over carbon graphite electrodes (CGE). As a mediator of the enzymatic product, Prussian blue (PB) was also electrodeposited over the working electrodes. The film provides electrochemical activity with a homogeneous coverage, and the use of PB does not alter the electrochemical profile or the film morphology. Analysis of infrared spectra demonstrates the presence of the unchanged carboxyl groups and suggests that polymerization begins at the nitrogen atom. The biosensor was developed on graphite screen-printed electrodes with all optimization performed on CGE. UOx was immobilized through physical adsorption and the proposed sensor was coupled to a flow cell in a flow injection analysis (FIA) system with amperometric detection. The flow rate, injection volume, and pH of the UA solution were optimized at 2.10 mL min − 1 , 200 μL, and 8.27, respectively. Under these conditions, a relative standard deviation of 2.15% (n = 10) was achieved for UA detection. A linear range of 10 to 200 μM was obtained for UA determination with a limit of detection of 3.0 μM. UA recovery from amended urine samples was 97.4% (± 2.4). [ABSTRACT FROM AUTHOR]

Raman spectroscopy can probe the structure and conformations of specific chemical groups within proteins and may thus be used as a technique complementary to X-ray crystallography. This combined approach can be decisive in resolving ambiguities in the interpretation of enzymatic or X-ray induced processes. Here, we present an online Raman setup developed at the European Synchrotron that allows for interleaved Raman spectra acquisition and X-ray diffraction measurements with fast probe exchange and simple alignment while maintaining a high sensitivity over the entire spectral range. This device has been recently employed in the study of a covalent intermediate in the O 2 -dependent breakdown of uric acid by the cofactor-free enzyme urate oxidase and to monitor its decay induced by X-ray exposure. [ABSTRACT FROM AUTHOR]

As human uricase has been silenced during evolution, counterparts from other species become an alternative for the treatment of hyperuricemia. Candida uricase is a promising option among them, but its aggregation propensity remains a major obstacle to clinical use. In this study, we designed two mutations according to homology-modeled 3D structure of Candida uricase: Cys249Ser substitution and C-terminal Leu deletion. The wild-type uricase and three mutants containing either or both of the mutations were expressed in Escherichia coli BL21 and validated by mass spectrometry. Size-exclusion chromatography and electrophoresis analysis demonstrated that aggregation was induced by interchain disulfide bonds and could be significantly avoided by Cys249Ser substitution. In combination with Cys249Ser substitution, deletion of Leu increased the enzymatic activity by 8%. Taken together, mutant containing both mutations is chosen as our target protein which is comparatively more suitable for therapeutic use. In addition, homology-modeled 3D structure was proved to be an efficient approach for protein engineering. [ABSTRACT FROM AUTHOR]

Objectives: Tophaceous gout seldom affects the axial skeleton. Symptoms vary according to the differential localization of urate deposits and the diagnosis is often delayed. Here, we report an unusual case of lumbar spinal stenosis caused by extradural tophaceous deposits.Methods: We retrospectively reviewed a case of a patient with tophaceous gout of the lumbar spine and reviewed the relevant literature.Results: A 62-year-old man with a 2-year history of lower back pain and a 3-month history of lower limb radiation pain and intermittent claudication was admitted. After laboratory and imaging investigations he underwent surgical decompression and stabilization. Histological analysis of the extracted specimen confirmed that it was gouty tophus. The patient's symptoms improved progressively after the operation. He recovered very well with no complications.Conclusion: The mechanism associated with axial gout is not yet clear. Obesity, inactivity, and previous degenerative disc disease may be the risk factors for spinal tophus. The clinical symptoms are diverse according to the differential localization of urate deposits. It is not easy to diagnose this disease radiographically by routine radiological examination. Analysis of a biopsy specimen is definitely the only way to confirm diagnosis. Surgical treatment should be considered in patients with spinal gout who are experiencing neurological deterioration. [ABSTRACT FROM AUTHOR]

The combination of methotrexate and pegloticase not only improves response rate by nearly double but also reduces infusion reactions by nearly eightfold. This is significant as a major drawback of using pegloticase monotherapy was the significant infusion reactions that made many practitioners unwilling to use the medication in patients with severe gout who may not have responded to conventional therapies. Those patients now have a chance to live a normal life free from gout. [ABSTRACT FROM AUTHOR]

Aspergillus flavus uricase (Rasburicase) with a molecular mass of 135 kDa is currently used for the treatment of gout and hyperuricemia occurring in tumor lysis syndrome. To characterize the effects of raffinose and lactose osmolytes on the uricase structure, its coding sequence was cloned, expressed in E. coli BL21, and purified by Ni-NTA agarose affinity chromatography. Thermal inactivation studies at 40 °C showed that nearly 15% of UOX activity was preserved, while the presence of raffinose and lactose reduced its activity to 35 and 45% of its original activity, respectively. Investigation of UOX thermal stability at 40 °C in the course of time showed that the enzyme relatively lost almost 60% of its original activity after 40 min, whereas more than 50% of UOX activity is preserved in the presence of lactose. Estimation of thermal inactivation rate constant, k , showed that the UOX k and UOX k in the presence of raffinose was unchanged (0.018 min), whereas for the presence of lactose, it was 0.015 min. Half-life and T analysis showed that UOX half-life is almost 38 min and addition of raffinose did not change the half-life, whereas the presence of lactose had remarkable impact on UOX half-life (46 min). The presence of raffinose increased UOX T to a lesser extent, whereas lactose notably enhanced the T from 27 to 37 °C. Overall, our findings show that lactose has protective effects on UOX stability, while for raffinose, it is relatively compromised. [ABSTRACT FROM AUTHOR]

Urate oxidase is a key enzyme in purine metabolism and catalyzes the oxidation of uric acid to allantoin. It is used to treat hyperuricemia and gout, and also in a diagnostic kit. In this study, error-prone polymerase chain reaction and staggered extension process was used to generate a mutant urate oxidase with improved enzyme activity from Bacillus subtilis. After several rounds of mutagenesis and screening, two mutants 6E9 and 8E279 were obtained which exhibited 2.99 and 3.43 times higher catalytic efficiency, respectively. They also exhibited lower optimal reaction temperature and higher thermo-stability. D44V, Q268R and K285Q were identified as the three most beneficial amino acid substitutions introduced by site-directed mutagenesis. D44V/Q268R, which was obtained through random combination of the three mutants, displayed the highest catalytic activity. The Km, kcat/Km and enzyme activity of D44V/Q268R increased by 68%, 83% and 129% respectively, compared with that of wild-type urate oxidase. Structural modeling indicated that mutations far from the active site can have significant effects on activity. For many of them, the underlying mechanisms are still difficult to explain from the static structural model. We also compared the effects of the same set of single point mutations on the wild type and on the final mutant. The results indicate strong effects of epistasis, which may imply that the mutations affect catalysis through influences on protein dynamics besides equilibrium structures. [ABSTRACT FROM AUTHOR]

This study explores the structural and functional changes associated with a low-temperature thermal transition of 2 engineered bacterial uricase mutants. Uricase has a noncovalent homotetrameric structure, with 4 active sites located at the interface of subunits. Using differential scanning calorimetry, a low-temperature transition was identified at 42°C for mutant A and at 33°C for mutant B. This transition was stabilized by the uricase inhibitor, oxonic acid, suggesting a strong structural relationship to the active site. For mutant B, there was a reversible loss of enzymatic activity above the low-temperature transition. Spectroscopic measurements demonstrated that there was also a reversible loss of secondary and tertiary structures and an increase in surface hydrophobicity. However, the hydrophobic core environment and the tetrameric structure were not altered over the low-temperature transition suggesting that the changes occurred primarily at the surface of the enzyme. The protein became aggregation-prone at temperatures approaching the cluster of higher-temperature melting transitions at 84°C, indicating these transitions represent a global unfolding of the protein. Our findings shed light on the structural changes that affect the uricase mechanism of action and provide new insights into how enzyme therapeutic development may be approached. [ABSTRACT FROM AUTHOR]

Lucilia sericata maggots are the only species currently approved for maggot debridement therapy (MDT), an alternative treatment for chronic and recalcitrant wounds. Maggots promote wound debridement, disinfection and healing by producing a complex mixture of proteins, peptides and low-molecular-weight compounds in their secretions and excretions, but the individual components are not well characterized at the molecular level. Here we investigated the purine catabolism pathway in L. sericata , focusing on the production of allantoin by Urate Oxidase (UO), which is thought to promote wound healing. We produced recombinant L. sericata UO in Escherichia coli , and characterized the properties of the pure enzyme in terms of the optimum pH (7–10) and temperature (20–25 °C), its stability, sensitivity to inhibition and ion dependency. We used quantitative RT-PCR and RNA in situ hybridization to monitor the expression of the UO gene, and we used a guinea pig anti-UO antibody to detect the native enzyme by western blot and by florescence immunohistochemistry in larval tissues. We found that L. sericata UO is exclusively present in the larval excretion organ (the Malpighian tubes) and is freely available in the cytoplasm rather than restricted to a specific subcellular compartment. Allantoin is a final product of L. sericata purine catabolism. It is produced by UO in the Malpighian tubes to remove uric acid from the hemolymph and is consequently excreted via the hindgut. Our findings confirm the hypothesis that both actively secreted molecules and excretion products contribute to the beneficial effects of MDT. [ABSTRACT FROM AUTHOR]

Uricase is a clinical enzyme used for the oxidation of uric acid crystals in gout disease. The present study aimed to increase the suitable surfactant-mediated uricase production on induction by different concentrations of inducers. The efficiency ofBacillus cereusto produce extracellular uricase enzyme was studied in uric acid-containing agar plates. Among the studied inducers, uric acid is the potential inducer for uricase production under submerged fermentations (SMF), which induced 19.41 U/ml uricase in medium containing 2.0 g/L of uric acid, however further increase in the uric acid concentration decreased uricase production, which could be because of substrate inhibition. The physical parameters including agitation speed (rpm) and time duration (h) of uricase production were optimized and found to produce optimum uricase at 150 rpm in 26 h of SMF. Among the studied surfactants, nonionic surfactant, polyvinyl alcohol has shown a remarkable increase in the uricase production of 31.58 U/ml, which is a 61% increase under optimized conditions in SMF. The stability of produced uricase was found at pH 7.5 and temperature 30°C. Also the effects of various metal ions (1 mM) on the uricase activity were studied and observed to be inhibitory in nature in the descending order K+ > Ca2+ > Zn2+ > Fe3+ > Ni2+ > Mg2+ > Mn2+ > Cu2+. [ABSTRACT FROM PUBLISHER]

Urate oxidase (Uox) has significant properties for assessment of serum uric acid and its kits are generally used, unfortunately, most of the third world countries are totally relay on the import of the said kit. The industrialisation of said kit in Pakistan is our need. Hence, this work is a part of a project in which stabilised hyper produced Uox (68.05 ± 3.50 U/mg, pH 8.5, temperature 35°C) was utilised for preparation of diagnostic kit. Peroxidase was also used in kit for improving the specificity of the kit. Finally the self-prepared kit was compared with two standard kits and it was found that there was no significant difference between the self-prepared kit and standard kits. It was also found that glucose (4-5 g/l), aspirin (0.5-1 g/l), glutathione (0.5-1 g/l), protein (40-60 g/l) did not interfere in determination of uric acid while heparin (100-200 mg/dl), urea (50-100 mg/dl), creatinine (10-20 mg/dl), bilirubin (10-20 mg/dl) and ascorbic acid (10-20 mg/l) interfere. [ABSTRACT FROM AUTHOR]

Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout. [ABSTRACT FROM AUTHOR]

In this work, a novel and simple fluorescence method for detection of uric acid (UA) based on NaYF 4 :Yb 3+ , Tm 3+ upconversion nanoparticles (UCNPs) is developed. The proposed method is based on the fact that uricase can oxidize uric acid to allantoin and hydrogen peroxide, which, on its turn, can oxidize o-phenylenediamine (OPD) to the oxidized OPD (oxOPD). The fluorescence of UCNPs can be significantly quenched by oxOPD through inner filter effects (IFE). Under the optimized conditions, the UA concentration was proportional to the changes in fluorescence intensity of UCNPs. A linear response was obtained over the concentration range from 20 to 850 μΜ with the low detection limit of 6.7 μΜ for uric acid. More importantly, this method has the potential to detect uric acid in human serum samples, suggesting the nanosensor can be used in a complex biological sample matrix. [ABSTRACT FROM AUTHOR]

Mitochondria play a critical role in energy production, cell signalling and cell survival. Defects in mitochondrial function contribute to the ageing process and ageing-related disorders such as metabolic disease, cancer, and neurodegeneration. The sirtuin family of deacylase enzymes have a variety of subcellular localisations and have been found to remove a growing list of post-translational acyl modifications from target proteins. SIRT3, SIRT4, and SIRT5 are found primarily located in the mitochondria, and are involved in many of the key processes of this organelle. SIRT3 has been the subject of intense research and is primarily a deacetylase thought to function as a mitochondrial fidelity protein, with roles in mitochondrial substrate metabolism, protection against oxidative stress, and cell survival pathways. Less is known about the functional targets of SIRT4, which has deacetylase, ADP-ribosylase, and a newly-described lipoamidase function, although key roles in lipid and glutamine metabolism have been reported. SIRT5 modulates a host of newly-discovered acyl modifications including succinylation, malonylation, and glutarylation in both mitochondrial and extra-mitochondrial compartments, however the functional significance of SIRT5 in the regulation of many of its proposed target proteins remains to be discovered. Because of their influence on a broad range of pathways, SIRT3, SIRT4, and SIRT5 are implicated in a range of disease-states including metabolic disease such as diabetes, neurodegenerative diseases, cancer, and ageing-related disorders such as hearing-loss and cardiac dysfunction. We review the current knowledge on the function of the three mitochondrial sirtuins, their role in disease, and the current outstanding questions in the field. [ABSTRACT FROM AUTHOR]

Uric acid (UA) is a purine metabolite that in most mammals is degraded by the hepatic enzyme uricase to allantoin. Epidemiological studies have shown that an elevated UA level predicts the development of cognition and memory deficits; however, there is no direct evidence of this relationship, and the underlying mechanism is largely undefined. Here, we show that a high-UA diet triggers the expression of proinflammatory cytokines, activates the Toll-like receptor 4 (TLR4)/nuclear factor (NF)-κB pathway, and increases gliosis in the hippocampus of Wistar rats. We, subsequently, identify a specific inhibitor of NF-κB, BAY11-7085, and show that stereotactic injections of the inhibitor markedly ameliorate UA-induced hippocampal inflammation and memory deficits in C57BL/6 mice. We also found that NF-κB is activated in the primary cultured hippocampal cells after UA administration. Additionally, C57BL/6 mice that lack TLR4 are substantially protected against UA-induced cognitive dysfunction, possibly due to a decrease in inflammatory gene expression in the hippocampus. Importantly, magnetic resonance imaging confirms that hyperuricemia in rats and humans is associated with gliosis in the hippocampus. Together, these results suggest that UA can cause hippocampal inflammation via the TLR4/NF-κB pathway, resulting in cognitive dysfunction. Our findings provide a potential therapeutic strategy for counteracting UA-induced neurodegeneration. [ABSTRACT FROM AUTHOR]

Although uricase-knockout (Uox KO) mice are reported to develop uric acid (UA) nephropathy, those that mature without severe nephropathy could be useful for research into purine metabolism in humans. In this study, we measured the urinary excretion of creatinine, UA, allantoin, and 8-hydroxy-2′-deoxyguanosine (8-OHdG) collected from Uox KO mice housed in metabolic cages. UA and allantoin were determined using liquid chromatography–mass spectrometry and creatinine and 8-OHdG were measured with a commercial kit. Uox KO mice excreted significantly higher levels of UA than wild-type mice (C57BL/6), while the excretion of allantoin was significantly lower. Urinary allantoin was detected in Uox KO mice despite a lack of uricase, which is the same as in humans. In contrast to the elevated levels of UA, the daily excretion of 8-OHdG, an oxidative stress marker, was lower in Uox KO mice. UA is thought to act as an anti-oxidizing agent in humans; thus, these results show that Uox KO mice are potential animal models for research into human purine metabolism. [ABSTRACT FROM AUTHOR]