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Uricase is potential therapeutic and diagnostic enzyme having wide applications in pharmaceutical sector, but production yield and cost of production are challenging. Hence, isolation of potent uricase-producing sources and optimizing its production are essential for industrial-scale uricase production. In this study, a novel extracellular uricase-producing bacterium was isolated using uric acid plate assay. The strain was identified by 16S rRNA sequencing and it was found to be Pseudomonas mosselii with similarity of 99%. Further, multi-level statistical optimizations were conducted to enhance the uricase production. Firstly, Plackett-Burman design (PBD) was adapted for screening of different medium components and found that uric acid, peptone, and sucrose are highly significant medium components which affect the uricase production. Further, central composite design (CCD) and artificial neural network (ANN) combined genetic algorithm (GA) were used to optimize the uricase production. The use of CCD prior to ANN improved the predictive capability of ANN and GA model. Under the optimum conditions obtained using ANN-GA, the uricase production was enhanced and found to be 59.91 U/mL ± 0.32 which is 2.02 folds higher compared to unoptimized medium. The results of the current study enunciate that the newly isolated Pseudomonas mosselii is a potential bacterium for enhanced production of uricase and optimization using multilevel optimization approaches gave better prediction of optimum process conditions for enhanced uricase production. [ABSTRACT FROM AUTHOR]

AbstractThis study aimed to identify proteolysis-resistant extracellular uricase by screening three hundred marine microorganisms, including bacteria, actinobacteria and filamentous fungi. The isolate, demonstrating resistance to proteolysis and producing extracellular uricase with relatively high activity was identified as Streptomyces rochei 6SM85. The enzyme was purified approximately 3.6 fold, yielding 1.23%, using ion exchange chromatography. The molecular weight of the purified uricase was estimated to be approximately 14 kDa by SDS-PAGE. The optimum temperature and pH for uricase were determined to be 35 °C and 8–9, respectively. It was demonstrated that uricase retained up to 46% of its activity after half an hour at 40 °C and exhibited higher activity at neutral and basic pH compared to acidic pH. Its activity increased in the presence of 1 mM Mg2+, Na+, and EDTA, while it decreased by 86% in the presence of Cu2+. This study represents the first report on the production and characterization of extracellular proteolysis-resistant uricase from marine-derived Streptomyces. [ABSTRACT FROM AUTHOR]

Improving the activity of uricase and lowering its immunogenicity remain significant challenges in the enzyme replacement management of hyperuricemia and related inflammatory diseases. Herein, an immunogenicity-masking strategy based on engineered red blood cells (RBCs) was developed for effective uricase delivery against both hyperuricemia and gout. The dynamic membrane of RBCs enabled high resistance to protease inactivation and hydrogen peroxide accumulation. Benefiting from these advantages, a single infusion of RBC-loaded uricase (Uri@RBC) performed prolonged blood circulation and sustained hyperuricemia management. Importantly, RBCs masked the immunogenicity of uricase, leading to the maintenance of UA-lowering performance after repeated infusion through reduced antibody-mediated macrophage clearance. In an acute gout model, Uri@RBC profoundly alleviated joint edema and inflammation with minimal systemic toxicity. This study supports the employment of immunogenicity-masking tools for efficient and safe enzyme delivery, and this strategy may be leveraged to improve the usefulness of enzyme replacement therapies for managing a wide range of inflammatory diseases. [Display omitted] [ABSTRACT FROM AUTHOR]

Background: The thyroid gland is one of the largest endocrine glands in the body, secreting thyroxine (T4) and triiodothyronine (T3). Mild hypothyroidism can have negative consequences for a variety of tissues, even though clinically the patients are euthyroid. Many research studies were conducted to evaluate the serum creatinine levels of people with hypothyroidism; however, relatively little research was conducted to evaluate serum urea and serum creatinine levels in subclinical hypothyroidism. As a result, the current study was carried out to investigate urea, uric acid, and creatinine levels in hypothyroidism. OBJECTIVES: Our study aimed to determine and correlate the levels of urea, uric acid and creatinine in newly diagnosed primary hypothyroidism cases. MATERIALS AND METHODS: In this cross-sectional study, 96 newly diagnosed cases of primary hypothyroidism were included. The findings were correlated with 20–60-year-old and sex-matched cases. Serum urea was determined by the kinetic method (Glutamate Dehydrogenase Method), serum uric acid by the uricase method and serum creatinine by Jaffe’s method in Erba’s Mannheim fully automated analyser. Result: This study showed a positive correlation between uric acid and thyroid stimulating hormone (TSH) and a negative correlation between urea and creatinine. There was no statistical significance in the mean values of urea, uric acid and creatinine but an increased level of urea, uric acid and creatinine was seen in the age group 20–30 years when compared with the other age groups. Conclusion: The study concludes that serum urea showed a positive correlation with TSH, whereas uric acid and creatinine showed a negative correlation with TSH, and there was no statistical significance in the mean values of urea, uric acid and creatinine in hypothyroid patients.

Hyper-uric acid (UA)-induced kidney injury (HAKI) is caused by the deposition of excess blood UA into the kidneys. We confined molecules of uricase (URI), catalase (CAT), and curcumin (Cur) to a single structure (UC/Cur) while retaining their enzymatic activities via a cross-linking complexation reaction between tannic acid and FeCl3 for treating HAKI. Simultaneously, bovine serum albumin (BSA)-UC/Cur nanoparticles were successfully prepared by interlinking the disulfide bonds of BSA with the enzyme complex via Tris(2-carboxyethyl) phosphine(TCEP) to form sulfhydryl groups. BSA-UC/Cur significantly attenuated MSU-induced NLRP3 inflammasome pathway activation and apoptosis in NRK-52e cells by eliminating UA crystals and intracellular reactive oxygen species. More importantly, treatment with BSA-UC/Cur stabilized blood UA concentrations and lowered proximal tubular protein levels, mitochondrial swelling, and fibrotic areas, renducing the expression of matrix metalloproteinase (MMP)2, MMP9, and NLRP3 while, increasing the expression of tight-junction proteins ZO1 and occludin as well as that of TIMP-1, in HAKI model rats. In addition, BSA-UC/Cur nanoparticles reduced the subpopulation ratios of CD8+ T cells and M1 macrophages and increased those of M2 macrophages and Treg cells. Preliminary in-vivo trials showed that long-term intravenous treatment with BSA-UC/Cur is safe. Therefore, BSA-UC/Cur could be a potential nanotherapeutic agent for HAKI.

The uricase enzyme yields allantoin, hydrogen peroxide, and carbon dioxide by catalyzing the oxidative opening of the purine ring in the urate pathway. This enzyme is important for biochemical diagnosis and reduces toxic urate accumulation during various diseases (hyperuricemia, gout, and bedwetting). Direct urate oxidase injection is recommended in renal complications-associated gout and to prevent chemotherapy-linked hyperuricemia disorders. Thus, uricase is a promising enzyme with diverse applications in medicine. Microbial production of uricase is featured by high growth rates, cost-effective bioprocessing, and easy optimization of the medium. Microbes produce the enzyme extracellular or intracellular. Extracellular uricase is preferred for biotechnological applications as it minimizes time, effort, and purification processes. This review provides insights into uricase-producing microbes, bacterial uric acid degradation pathways, degrading enzymes, and uricase-encoding genes.

Uric acid is the final metabolite in humans. High level of uric acid chronically induces urate deposition, aggravates kidney damage, and concomitantly causes an increase in inflammatory factors. Alleviating acute inflammation and decreasing uric acid levels are the key points in the treatment of inflammatory diseases associated with high uric acid. However, a drug delivery system that combines anti-inflammatory and uric acid reduction functions at the same time remains a challenge to be settled. Here, we designed a nanocrystal-based co-delivery platform, IND Nplex, characterized by loading of indomethacin (IND) and uricase. Compared with free IND or uricase, IND Nplex possessed a better anti-inflammatory effect by restraining the release of inflammation-related factors in vitro. In addition, pharmacokinetic and biodistribution studies revealed that IND Nplex significantly prolonged the retention time in vivo and was more concentrated in the kidney. In acute gouty arthritis model rats, IND Nplex markedly relieved ankle joint swelling and mitigated synovial inflammation. In acute kidney injury model rats, IND Nplex indicated better biocompatibility and significant amelioration of renal fibrosis. Moreover, IND Nplex showed the effect of anti-inflammatory and improved renal function via determination of inflammatory factors and biochemical markers in the serum and kidney. In conclusion, these results indicate that IND Nplex exerts anti-inflammatory activity and uric acid–lowering effect and could become a promising candidate for the treatment of uric acid–related diseases. In this study, a stable neutral co-delivery platform (IND Nplex) was fabricated by using a co-delivery strategy, allowing for a combination of the anti-inflammation drug IND and the metabolic enzyme uricase. IND Nplex showed a good therapeutic effect on rats with acute gouty arthritis or acute kidney injury due to the dual effects of anti-inflammatory and uric acid lowering. This platform provided a novel strategy for high uric acid–related illnesses. [ABSTRACT FROM AUTHOR]

Humans are predisposed to gout because they lack uricase that converts uric acid to allantoin. Rodents have uricase, resulting in low basal serum uric acid. A uricase inhibitor raises serum uric acid in rodents. There were two aims of the study in polycystic kidney disease (PKD): 1) to determine whether increasing serum uric acid with the uricase inhibitor, oxonic acid, resulted in faster cyst growth and 2) to determine whether treatment with the xanthine oxidase inhibitor, oxypurinol, reduced the cyst growth caused by oxonic acid. Orthologous models of human PKD were used: PCK rats, a polycystic kidney and hepatic disease 1 (Pkhd1) gene model of autosomal recessive PKD (ARPKD) and Pkd1RC/RC mice, a hypomorphic Pkd1 gene model. In PCK rats and Pkd1RC/RC mice, oxonic acid resulted in a significant increase in serum uric acid, kidney weight, and cyst index. Mechanisms of increased cyst growth that were investigated were proinflammatory cytokines, the inflammasome, and crystal deposition in the kidney. Oxonic acid resulted in an increase in proinflammatory cytokines in the serum and kidney in Pkd1RC/RC mice. Oxonic acid did not cause activation of the inflammasome or uric acid crystal deposition in the kidney. In Pkd1RC/RC male and female mice analyzed together, oxypurinol decreased the oxonic acid-induced increase in cyst index. In summary, increasing serum uric acid by inhibiting uricase with oxonic acid results in an increase in kidney weight and cyst index in PCK rats and Pkd1RC/RC mice. The effect is independent of inflammasome activation or crystal deposition in the kidney. NEW & NOTEWORTHY: This is the first reported study of uric acid measurements and xanthine oxidase inhibition in polycystic kidney disease (PKD) rodents. Raising serum uric acid with a uricase inhibitor resulted in increased kidney weight and cyst index in Pkd1RC/RC mice and PCK rats, elevated levels of proinflammatory cytokines in the serum and kidney in Pkd1RC/RC mice, and no uric acid crystal deposition or activation of the caspase-1 inflammasome in the kidney. [ABSTRACT FROM AUTHOR]

The uricase enzyme yields allantoin, hydrogen peroxide, and carbon dioxide by catalyzing the oxidative opening of the purine ring in the urate pathway. This enzyme is important for biochemical diagnosis and reduces toxic urate accumulation during various diseases (hyperuricemia, gout, and bedwetting). Direct urate oxidase injection is recommended in renal complications-associated gout and to prevent chemotherapy-linked hyperuricemia disorders. Thus, uricase is a promising enzyme with diverse applications in medicine. Microbial production of uricase is featured by high growth rates, cost-effective bioprocessing, and easy optimization of the medium. Microbes produce the enzyme extracellular or intracellular. Extracellular uricase is preferred for biotechnological applications as it minimizes time, effort, and purification processes. This review provides insights into uricase-producing microbes, bacterial uric acid degradation pathways, degrading enzymes, and uricase-encoding genes. Furthermore, aspects influencing the microorganisms' production of the uricase enzyme, its activity, and its purification procedure are also emphasized. Cell disruption is mandatory for intercellular uricase production, which elevates production costs. Therefore, extracellular uricase-producing microbial strains should be investigated, and production factors should be optimized. Future techniques for obtaining extracellular enzymes should feature reduced time and effort, as well as a simple purification methodology. Furthermore, uricase gene-carrying recombinant probiotic microorganisms could become an effective tool for gout treatment. [ABSTRACT FROM AUTHOR]

Objectives Serum urate (SU) lowering with PEGylated uricases in gout can reduce flares and tophi. However, treatment-emergent anti-drug antibodies adversely affect safety and efficacy and the currently approved PEGylated uricase pegloticase requires twice-monthly infusions. Investigational SEL-212 therapy aims to promote uricase-specific tolerance via monthly sequential infusions of a proprietary rapamycin-containing nanoparticle (ImmTOR) and pegadricase. Methods COMPARE was a randomized, phase 2, open-label trial of SEL-212 vs pegloticase in adults with refractory gout. SEL-212 [ImmTOR (0.15 mg/kg) and pegadricase (0.2 mg/kg)] was infused monthly or pegloticase (8 mg) twice monthly for 6 months. The primary endpoint was the proportion of participants with SU <6 mg/dl for ≥80% of the time during 3 and 6 months. Secondary outcomes were mean SU, gout flares, number of tender and/or swollen joints and safety. Results During months 3 and 6 combined, numerically more participants achieved and maintained a SU <6 mg/dl for ≥80% of the time with SEL-212 vs pegloticase (53.0% vs 46.0%, P  = 0.181). The percentage reductions in SU levels were statistically greater during months 3 and 6 with SEL-212 vs pegloticase (−73.79% and −47.96%, P  = 0.0161). Reductions in gout flare incidence and number of tender and/or swollen joints were comparable between treatments. There were numerical differences between the most common treatment-related adverse events of interest with SEL-212 and pegloticase: gout flares (60.2% vs 50.6%), infections (25.3% vs 18.4%) and infusion-related reactions (15.7% vs 11.5%), respectively. Stomatitis (and related terms) was experienced by eight participants (9.6%) with SEL-212 and none with pegloticase. Stomatitis, a known event for rapamycin, was associated with ImmTOR only. Conclusions SEL-212 efficacy and tolerability were comparable to pegloticase in refractory gout. This was associated with a substantial reduction in treatment burden with SEL-212 due to decreased infusion frequency vs pegloticase. Clinical trial registration NCT03905512. [ABSTRACT FROM AUTHOR]

Cardiorenal syndrome (CRS) involves joint dysfunction of the heart and kidney. Acute forms share biochemical alterations like hyperuricaemia (HU) with tumour lysis syndrome (TLS). The mainstay treatment of acute CRS with systemic overload is diuretics, but rasburicase is used in TLS to prevent and treat hyperuricaemia. An observational, retrospective study was performed to assess the effectiveness and safety of a single dose of rasburicase in hospitalized patients with cardiorenal syndrome, worsening renal function and uric acid levels above 9 mg/dL. Rasburicase improved diuresis and systemic congestion in the 35 patients included. A total of 86% of patients did not need to undergo RRT, and early withdrawal was possible in the remaining five. Creatinine (Cr) decreased after treatment with rasburicase from a peak of 3.6 ± 1.27 to 1.79 ± 0.83 mg/dL, and the estimated glomerular filtration rate (eGFR) improved from 17 ± 8 to 41 ± 20 mL/min/1.73 m2 (p = 0.0001). The levels of N-terminal type B Brain Natriuretic Peptide (Nt-ProBNP) and C-reactive protein (CRP) were also significantly reduced. No relevant adverse events were detected. Our results show that early treatment with a dose of rasburicase in patients with CRS and severe HU is effective to improve renal function and systemic congestion, avoiding the need for sustained extrarenal clearance, regardless of comorbidities and ventricular function. [ABSTRACT FROM AUTHOR]

A flow-through biosensor system for the determination of uric acid was developed on the platform of flow-through electrochemical cell manufactured by 3D printing from poly(lactic acid) and equipped with a modified screen-printed graphite electrode (SPE). Uricase was immobilized to the inner surface of a replaceable reactor chamber. Its working volume was reduced to 10 μL against a previously reported similar cell. SPE was modified independently of the enzyme reactor with carbon black, pillar[5]arene, poly(amidoamine) dendrimers based on the p-tert-butylthiacalix[4]arene (PAMAM-calix-dendrimers) platform and electropolymerized 3,7-bis(4-aminophenylamino) phenothiazin-5-ium chloride. Introduction of the PAMAM-calix-dendrimers into the electrode coating led to a fivefold increase in the redox currents of the electroactive polymer. It was found that higher generations of the PAMAM-calix-dendrimers led to a greater increase in the currents measured. Coatings consisted of products of the electropolymerization of the phenothiazine with implemented pillar[5]arene and PAMAM-calix-dendrimers showing high efficiency in the electrochemical reduction of hydrogen peroxide that was formed in the enzymatic oxidation of uric acid. The presence of PAMAM-calix-dendrimer G2 in the coating increased the redox signal related to the uric acid assay by more than 1.5 times. The biosensor system was successfully applied for the enzymatic determination of uric acid in chronoamperometric mode. The following optimal parameters for the chronoamperometric determination of uric acid in flow-through conditions were established: pH 8.0, flow rate 0.2 mL·min−1, 5 U of uricase per reactor. Under these conditions, the biosensor system made it possible to determine from 10 nM to 20 μM of uric acid with the limit of detection (LOD) of 4 nM. Glucose (up to 1 mM), dopamine (up to 0.5 mM), and ascorbic acid (up to 50 μM) did not affect the signal of the biosensor toward uric acid. The biosensor was tested on spiked artificial urine samples, and showed 101% recovery for tenfold diluted samples. The ease of assembly of the flow cell and the low cost of the replacement parts make for a promising future application of the biosensor system in routine clinical analyses. [ABSTRACT FROM AUTHOR]

Despite growing prevalence and incidence, the management of gout remains suboptimal. The intermittent nature of the gout makes the long-term urate-lowering therapy (ULT) particularly important for gout management. However, patients are reluctant to take medication day after day to manage incurable occasional gout flares, and suffer from possible long-term toxicity. Therefore, a safe and easy-to-operate drug delivery system with simple preparation for the long-term management of gout is very necessary. Here, a chitosan-containing sustained-release microneedle system co-loaded with colchicine and uricase liposomes were fabricated to achieve this goal. This microneedle system was confirmed to successfully deliver the drug to the skin and maintain a one-week drug retention. Furthermore, its powerful therapeutic potency to manage gout was investigated in both acute gouty and chronic gouty models. Besides, the drug co-delivery system could help avoid long-term daily oral colchicine, a drug with a narrow therapeutic index. This system also avoids mass injection of uricase by improving its stability, enhancing the clinical application value of uricase. In general, this two-drug system reduces the dosage of uricase and colchicine and improves the patient's compliance, which has a strong clinical translation.

In this narrative review, we present the hypothesis that key mutations in two genes, occurring 15 and 10 million years ago (MYA), were individually and then collectively adaptive for ancestral humans during periods of starvation, but are maladaptive in modern civilization (i.e., "thrifty genes"), with the consequence that these genes not only increase our risk today for obesity, but also for alcoholism. Both mutations occurred when ancestral apes were experiencing loss of fruit availability during periods of profound climate change or environmental upheaval. The silencing of uricase (urate oxidase) activity 15 MYA enhanced survival by increasing the ability for fructose present in dwindling fruit to be stored as fat, a consequence of enhanced uric acid production during fructose metabolism that stimulated lipogenesis and blocked fatty acid oxidation. Likewise, a mutation in class IV alcohol dehydrogenase ~10 MYA resulted in a remarkable 40-fold increase in the capacity to oxidize ethanol (EtOH), which allowed our ancestors to ingest fallen, fermenting fruit. In turn, the EtOH ingested could activate aldose reductase that stimulates the conversion of glucose to fructose, while uric acid produced during EtOH metabolism could further enhance fructose production and metabolism. By aiding survival, these mutations would have allowed our ancestors to generate more fat, primarily from fructose, to survive changing habitats due to the Middle Miocene disruption and also during the late-Miocene aridification of East Africa. Unfortunately, the enhanced ability to metabolize and utilize EtOH may now be acting to increase our risk for alcoholism, which may be yet another consequence of once-adaptive thrifty genes.

A wearable biosensor was specifically engineered to measure uric acid, a biomarker present at wound sites. This biosensor, fabricated as a disposable and wearable device, was seamlessly integrated onto a polyethylene terephthalate (PET) substrate by utilizing carbon and silver conductive paste as the electrodes. The enzyme uricase was immobilized onto the working electrode by utilizing chitosan, a biocompatible material, to create this biosensor. Notably, the uric acid biosensor fabricated with chitosan showcased exceptional performance metrics, including remarkable output current values and impeccable stability. These findings suggest the prospective utilization of chitosan-based uric acid biosensors for the accurate measurement of uric acid on human skin in future applications. [ABSTRACT FROM AUTHOR]

In recent years, the development of electrochemical biosensors for uric acid has made great achievements. Firstly, uric acid electrochemical biosensors were classified according to their reaction mechanism. Then, the reaction mechanism of the uric acid sensor and the application of nano‐modified materials were deeply analyzed from the perspective of non‐enzyme and enzymes. In this paper, the catalytic oxidation capacity, enzyme adsorption effect, conductivity, robustness, detection range, and detection limit of uric acid sensors were discussed and compared. Finally, the advantages of acid‐sensitive electrochemical biosensors were summarized, and the constructive recommendations were proposed for improving the deficiencies of acid biosensors. The potential for further development in this area was also discussed. [ABSTRACT FROM AUTHOR]

The past year generated significant change and advancement of the urate field with novel insights regarding the role of uric acid (UA) in multiple pathophysiologic processes from gout to COVID-19. While these contributions continue to move the field forward, the basic biochemistry and biology of UA is often overlooked, being lost in the shadow of clinical associations and omics. However, the seminal impact of UA begins with biochemistry and the associated interplay with cell biology. In these basic reactions and resultant impacts on physiology, UA mediates its influence on clinical outcomes. As such, this review focuses on published advances in UA biochemistry and biology in 2022 and associates these advances with downstream consequences. [ABSTRACT FROM AUTHOR]

Uric acid is the main means of nitrogen excretion in uricotelic vertebrates (birds and reptiles) and the end product of purine catabolism in humans and a few other mammals. While uricase is inactivated in mammals unable to degrade urate, the presence of orthologous genes without inactivating mutations in avian and reptilian genomes is unexplained. Here we show that the Gallus gallus gene we name cysteine-rich urate oxidase (CRUOX) encodes a functional protein representing a unique case of cysteine enrichment in the evolution of vertebrate orthologous genes. CRUOX retains the ability to catalyze urate oxidation to hydrogen peroxide and 5-hydroxyisourate (HIU), albeit with a 100-fold reduced efficiency. However, differently from all uricases hitherto characterized, it can also facilitate urate regeneration from HIU, a catalytic property that we propose depends on its enrichment in cysteine residues. X-ray structural analysis highlights differences in the active site compared to known orthologs and suggests a mechanism for cysteine-mediated self-aggregation under H2O2-oxidative conditions. Cysteine enrichment was concurrent with the transition to uricotelism and a shift in gene expression from the liver to the skin where CRUOX is co-expressed with β-keratins. Therefore, the loss of urate degradation in amniotes has followed opposite evolutionary trajectories: while uricase has been eliminated by pseudogenization in some mammals, it has been repurposed as a redox-sensitive enzyme in the reptilian skin. [ABSTRACT FROM AUTHOR]

The present study aims to immobilize the uricase enzyme on magnetic nanowires and to examine its potential for use in the treatment of gout. For this, Au/Ni/Au nanowires were synthesized using a polycarbonate membrane template by the sequential electrodeposition of Au, Ni, and Au, respectively. The uricase enzyme was covalently attached to these nanowires and was also coated with PEG. Optimum enzymatic conditions, kinetic parameters, thermal, storage, and operational stability were determined by performing enzymatic activity tests of free and immobilized uricase. Additionally, the efficacy of both enzyme preparations in artificial human serum and the presence of protease was also investigated. Experimental results showed that immobilized uricase showed higher stability than free uricase in all studied conditions. The potential of immobilized uricase to oxidize uric acid in artificial serum was also investigated and it was found that immobilized preparation demonstrated approximately 6 times higher activity than that of the free enzyme. The results of this study showed that uricase-attached nanowires oxidized uric acid effectively and are promising in the treatment of gout. • Uricase enzyme was immobilized on the magnetic Au/Ni/Au nanowires. • The potential of uricase immobilized nanowire was examined to treat gout. • Immobilized uricase showed higher stability than free uricase. • Uricase-immobilized nanowires oxidized uric acid effectively. • These nanowires are promising in the treatment of gout. [ABSTRACT FROM AUTHOR]

In this research, for the first time, A. flavus uricase gene was cloned in pPink-UOX plasmid under strong alcohol oxidase promoter of Pichia pink expression system after codon optimization. After selecting the best uricase producing clone with an activity of 0.7 U/ml at the Flask level, a 5-L fermenter was used to increase the expression of the enzyme. Within 60 h, the fermentation process produced 1500 g of biomass from 4 L of semi defined culture media and expressed 2.5 g/L of the enzyme. The purity of recombinant uricase production using three consecutive DEAE Sepharose, CM Sepharose and Phenyl Sepharose columns was above 99%, which was confirmed by SDS-PAGE and RP-HPLC analyses. Size exclusion chromatography analysis showed that the purified enzyme has comparable heterogeneity to the Rasburicase. The yield of recombinant uricase production in this study was 63% and its specific activity was 24 U/mg. The high expression of recombinant uricase in the Pichia pink strain and the increased enzyme activity compared to the standard sample indicate the potential of therapeutic and diagnostic applications of recombinant uricase in the present study. [ABSTRACT FROM AUTHOR]

Uricase suppresses the harmful effects of uric acid accumulation by the degradation of uric acid to allantoin. Maintaining enzyme stability is a major challenge. The stability issue can be avoided when using enzymes loaded into polymeric structures. For this purpose, uricase loaded into responsive nanopolymers (UO-RNPs) was synthesized. The characterizations such as SEM, FTIR, and zeta potential of UO-RNPs were performed. Maximum uricase loading to RNPs was investigated and the release performance of UO-RNPs also under variable conditions such as pH and temperature. On the other hand, loading yield (LY) and loading efficiency (LE) was founded 73.22 ± 3.62% and 71.24 ± 4.61%, while storage stability and reusability of the UO-RNPs were found to be about 68% and 51% of the original activity after 4 weeks and 10 cycles, respectively. From Lineweaver-Burk plot the Km was founded 0.192 and 0.327 mM for loaded and free uricase. While V max was founded 0.131 and 0.0714 μM/min for free uricase and UO-RNPs, respectively. UO-RNPs showed promising matrices for high catalytic efficiency and enhanced stability. Up until now, as far as we know, the present study of the loaded uricase into responsive nanopolymers is the first attempt of uricase enzyme in such an investigation. [Display omitted] • The double responsive nanopolymers were synthesized for urease loading. • Uricase loading yield was found to be 73.22 ± 3.62%. • The activity was found above 51% after 10 repeated times for reusability. • The Km and V max were found as 0.192 mM and 0.0714 μmol/min for UO-RNPs. [ABSTRACT FROM AUTHOR]

Despite growing prevalence and incidence, the management of gout remains suboptimal. The intermittent nature of the gout makes the long-term urate-lowering therapy (ULT) particularly important for gout management. However, patients are reluctant to take medication day after day to manage incurable occasional gout flares, and suffer from possible long-term toxicity. Therefore, a safe and easy-to-operate drug delivery system with simple preparation for the long-term management of gout is very necessary. Here, a chitosan-containing sustained-release microneedle system co-loaded with colchicine and uricase liposomes were fabricated to achieve this goal. This microneedle system was confirmed to successfully deliver the drug to the skin and maintain a one-week drug retention. Furthermore, its powerful therapeutic potency to manage gout was investigated in both acute gouty and chronic gouty models. Besides, the drug co-delivery system could help avoid long-term daily oral colchicine, a drug with a narrow therapeutic index. This system also avoids mass injection of uricase by improving its stability, enhancing the clinical application value of uricase. In general, this two-drug system reduces the dosage of uricase and colchicine and improves the patient's compliance, which has a strong clinical translation. A chitosan-containing sustained-release microneedle system co-loaded with colchicine (Col) and uricase (UAO) liposomes was designed and fabricated to provide a simple and effective way for gout management. [Display omitted] [ABSTRACT FROM AUTHOR]

Cardiorenal syndrome (CRS) involves joint dysfunction of the heart and kidney. Acute forms share biochemical alterations like hyperuricaemia (HU) with tumour lysis syndrome (TLS). The mainstay treatment of acute CRS with systemic overload is diuretics, but rasburicase is used in TLS to prevent and treat hyperuricaemia. An observational, retrospective study was performed to assess the effectiveness and safety of a single dose of rasburicase in hospitalized patients with cardiorenal syndrome, worsening renal function and uric acid levels above 9 mg/dL. Rasburicase improved diuresis and systemic congestion in the 35 patients included. A total of 86% of patients did not need to undergo RRT, and early withdrawal was possible in the remaining five. Creatinine (Cr) decreased after treatment with rasburicase from a peak of 3.6 ± 1.27 to 1.79 ± 0.83 mg/dL, and the estimated glomerular filtration rate (eGFR) improved from 17 ± 8 to 41 ± 20 mL/min/1.73 m2 (p = 0.0001). The levels of N-terminal type B Brain Natriuretic Peptide (Nt-ProBNP) and C-reactive protein (CRP) were also significantly reduced. No relevant adverse events were detected. Our results show that early treatment with a dose of rasburicase in patients with CRS and severe HU is effective to improve renal function and systemic congestion, avoiding the need for sustained extrarenal clearance, regardless of comorbidities and ventricular function.

A flow-through biosensor system for the determination of uric acid was developed on the platform of flow-through electrochemical cell manufactured by 3D printing from poly(lactic acid) and equipped with a modified screen-printed graphite electrode (SPE). Uricase was immobilized to the inner surface of a replaceable reactor chamber. Its working volume was reduced to 10 μL against a previously reported similar cell. SPE was modified independently of the enzyme reactor with carbon black, pillar[5]arene, poly(amidoamine) dendrimers based on the p-tert-butylthiacalix[4]arene (PAMAM-calix-dendrimers) platform and electropolymerized 3,7-bis(4-aminophenylamino) phenothiazin-5-ium chloride. Introduction of the PAMAM-calix-dendrimers into the electrode coating led to a fivefold increase in the redox currents of the electroactive polymer. It was found that higher generations of the PAMAM-calix-dendrimers led to a greater increase in the currents measured. Coatings consisted of products of the electropolymerization of the phenothiazine with implemented pillar[5]arene and PAMAM-calix-dendrimers showing high efficiency in the electrochemical reduction of hydrogen peroxide that was formed in the enzymatic oxidation of uric acid. The presence of PAMAM-calix-dendrimer G2 in the coating increased the redox signal related to the uric acid assay by more than 1.5 times. The biosensor system was successfully applied for the enzymatic determination of uric acid in chronoamperometric mode. The following optimal parameters for the chronoamperometric determination of uric acid in flow-through conditions were established: pH 8.0, flow rate 0.2 mL·min−1, 5 U of uricase per reactor. Under these conditions, the biosensor system made it possible to determine from 10 nM to 20 μM of uric acid with the limit of detection (LOD) of 4 nM. Glucose (up to 1 mM), dopamine (up to 0.5 mM), and ascorbic acid (up to 50 μM) did not affect the signal of the biosensor toward uric acid. The biosensor was tested on spiked artificial urine samples, and showed 101% recovery for tenfold diluted samples. The ease of assembly of the flow cell and the low cost of the replacement parts make for a promising future application of the biosensor system in routine clinical analyses.

Gout is a form of inflammatory arthritis that results from elevated serum uric acid levels and the deposition of urate crystals in multiple joints. The inflammatory response during an acute gout attack is mediated by the activation of the NLRP3 inflammasome, leading to the release of IL-1β and inducing a localized tissue inflammatory response. Urate lowering therapies such as Pegloticase effectively reduce serum uric acid levels but are generally associated with an increase in acute gout flares. In this study, we developed a long-acting anti-inflammatory recombinant uricase by sequential fusing interleukin-1 receptor antagonist (IL-1Ra) and albumin-binding domain (ABD) with the N-terminal end of Arthrobacter globiformis uricase (AgUox). The recombinant uricase has longer in vivo half-life, and significantly alleviates monosodium urate (MSU) crystals induced inflammation in mouse model compared with the wild-type AgUox. This long-acting anti-inflammatory recombinant uricase has the potential to be developed as an effective urate lowering therapy with better safety profiles. • We report the first anti-inflammatory recombinant uricase. • Albumin-binding domain significantly prolongs in vivo half-life of recombinant uricase. • The dual-functional recombinant uricase has both urate-lowering and anti-inflammatory effects. [ABSTRACT FROM AUTHOR]

Five mini proteins mimicking uricase comprising 20, 40, 60, 80, and 100 amino acids were designed based on the conserved active site residues within the same dimer, using the crystal structure of tetrameric uricase from Arthrobacter globiformis (PDB ID: 2yzb) as a template. Based on molecular docking analysis, the smallest mini protein, mp20, shared similar residues to that of native uricase that formed hydrogen bonds with uric acid and was chosen for further studies. Although purified recombinant mp20 did not exhibit uricase activity, it showed specific binding towards uric acid and evinced excellent thermotolerance and structural stability at temperatures ranging from 10°C to 100°C, emulating its natural origin. To explore the potential of mp20 as a bioreceptor in uric acid sensing, mp20 was encapsulated within zeolitic imidazolate framework‐8 (mp20@ZIF‐8) followed by the modification on rGO‐screen printed electrode (rGO/SPCE) to maintain the structural stability. An irreversible anodic peak and increased semicircular arcs of the Nyquist plot with an increase of the analyte concentrations were observed by utilizing cyclic voltammetry and electrochemical impedance spectroscopy (EIS), suggesting the detection of uric acid occurred, which is based on substrate–mp20 interaction. [ABSTRACT FROM AUTHOR]

Miniaturized and integrated devices for fast determination of clinical biomarkers are in high demand in the current healthcare environment. In this work, we present a functionalized self-assembled monolayer (SAM) on the gold surface of a screen-printed electrode (Au-SPE). The device was applied for uric acid (UA) detection, a biomarker associated with arthritis, diabetes mellitus, and kidney function. Prior to SAM formation, AuSPE was subjected to pretreatment with KOH and Au electrodeposition to provide additional roughness to the substrate. The SAM was formed in the AuSPE/KOH/AuNP surface by the cysteamine method—carried out for working surface dipping in the cysteamine (CYS) solution at 20 mM for 24 h (rinsed with ethanol and milli-Q water). Then, the uricase enzyme was immobilized through physical absorption at room temperature for 1 h to obtain the AuSPE/KOH/AuNPs/SAM/Uox biosensor. The physical and electrochemical characterization of AuSPE modification was carried out by scanning electron microscopy (SEM) and cyclic voltammetry (CV). The calibrated data of the Au/KOH/AuNPs/SAM/Uox biosensor showed a linear relation in the range of 50–1000 µM, a sensibility of 0.1449 µA/[(µM)cm2], and a limit of detection (LOD) of 4.4669 µM. The Au/KOH/AuNPs/SAM/Uox also exhibited good selectivity for UA in the presence of ascorbic acid. Moreover, the methodology showed good reproducibility, stability, and sensitive detection of UA. This performance of the proposed biosensor is in good accordance with clinical needs and can be compared with previous biosensors based on nanostructured surfaces of high-fabrication complexity. [ABSTRACT FROM AUTHOR]

Herein, we describe a uricase immobilized Au/Ni/PANI microrocket for the motion-based potential hyperuricemia therapy platform. This study has aimed to investigate the use of uricase immobilized magnetically guided microrockets to lower the uric acid level. The outermost Au layer of the microrocket facilitates the immobilization of the uricase enzyme, while the Ni layer allows controlling the motion of the microrocket via an external magnetic field. The innermost layer of PANI was integrated to prevent Ni from leaking. Optimum parameters for enzymatic reactions were determined, and optimum pH for free and immobilized uricase was found to be as 8.5, while optimum temperature was 35 °C for both uricase preparations. Also, the kinetic parameters of both free and immobilized uricase were evaluated. Km value for free uricase was found to be as 0.1212 mM, while immobilized uricase demonstrated lower Km value of 0.017 mM. Vmax of free and immobilized uricase was found to be as 0.8613 µmol/min and 0.02685 µmol/min, respectively. The immobilization process caused uricase to exhibit advanced stability for medium pH, resistance against protease enzymes, improved storage stability, and reusability. It was also observed that the substrate affinity for uric acid increased significantly after immobilization. As a model experiment, activity experiments were performed in an artificial serum medium to demonstrate the potential of the proposed system for future in vivo use. Artificial tests indicated that uricase-modified microrockets showed higher enzymatic activity than that of free counterpart and enzyme-free control studies. [ABSTRACT FROM AUTHOR]

We reported on uric acid (UA) detection using a new fluorescence-based assay: poly-(vinylpyrrolidone) templated copper nanoclusters (PVP-CuNCs) with uricase in an aqueous medium, such as human urine with uricase. These nanoclusters were synthesized in a simple wet chemical method and their morphological and optical properties were examined with the aid of high-resolution transmission electron microscopy and optical absorbance/emission spectroscopy. The PVP-CuNCs acted as the fluorescence indicators that used the enzyme-catalyzed oxidation of UA with uricase. Adding UA into the hybrid PVP-CuNCs/uricase solution caused enzyme-catalyzed oxidation to occur, producing hydrogen peroxide (H2O2), allantoin, and carbon dioxide. The fluorescence intensity of PVP-CuNCs is decreased by this biocatalytically generated H2O2, and this decrease is proportional to the UA level. A calibration plot showed the linear relationship with the negative slope between fluorescence intensity and UA in the range of 5–100 × 10−7 mol/L. The limit of detection (LOD) of UA was estimated as 113 × 10−9 mol/L. This fluorescent probe turned out to be highly specific for UA over other biologically relevant molecules. The demonstrated capability of the PVP-CuNCs as the nanoprobes for quantification of the UA levels in human urine samples could potentially pave the way toward medical applications where a super-sensitive, cost-effective, and UA-specific diagnosis was required. [ABSTRACT FROM AUTHOR]

Caste-based reproductive division of labour in social insects is built on asymmetries in resource allocation within colonies. Kings and queens dominantly consume limited resources for reproduction, while non-reproductive castes such as workers and soldiers help reproductive castes. Studying the regulation of such asymmetries in resource allocation is crucial for understanding the maintenance of sociality in insects, although the molecular background is poorly understood. We focused on uric acid, which is reserved and used as a valuable nitrogen source in wood-eating termites. We found that king- and queen-specific degradation of uric acid contributes to reproduction in the subterranean termite Reticulitermes speratus. The urate oxidase gene (RsUAOX), which catalyses the first step of nitrogen recycling from stored uric acid, was highly expressed in mature kings and queens, and upregulated with differentiation into neotenic kings/queens. Suppression of uric acid degradation decreased the number of eggs laid per queen. Uric acid was shown to be provided by workers to reproductive castes. Our results suggest that the capacity to use nitrogen, which is essential for the protein synthesis required for reproduction, maintains colony cohesion expressed as the reproductive monopoly held by kings and queens. [ABSTRACT FROM AUTHOR]

Nanozymes have advantages over natural enzymes in terms of efficiency, stability, and economy. MVSM (Mixed Valence State MOF) is a nano‐oxidase with uricase‐like activity that may catalyze uric acid (UA) in the body into allantoin and H2O2 to treat gout and hyperuricemia by substituting natural uricase. However, it cannot specifically identify and choose UA. To increase the selectivity and affinity of MVSM for UA, the composite material MVSM@MIP is innovatively synthesized using a new synthetic approach termed the "two‐step synthesis method," which may prevent UA from being oxidized by MVSM during manufacture in this study. At the same time, this study also provides experimental proof of the effective creation of the material, the advantages of the "two‐step synthesis approach," and the high selectivity and affinity of MVSM@MIP for UA. Based on these findings, the suggested technique may be used to effectively catalyze uric acid in human urine with high activity. [ABSTRACT FROM AUTHOR]

Gout is caused by elevated serum urate leading to the deposition of monosodium urate (MSU) crystals that can trigger episodes of acute inflammation. Humans are sensitive to developing gout because they lack a functional urate-metabolizing enzyme called uricase/urate oxidase (encoded by the UOX gene). A hallmark of long-standing disease is tophaceous gout, characterized by the formation of tissue-damaging granuloma-like structures ('tophi') composed of densely packed MSU crystals and immune cells. Little is known about how tophi form, largely due to the lack of suitable animal models in which the host response to MSU crystals can be studied in vivo long-term. We have previously described a larval zebrafish model of acute gouty inflammation where the host response to microinjected MSU crystals can be live imaged within an intact animal. Although useful for modeling acute inflammation, crystals are rapidly cleared following a robust innate immune response, precluding analysis at later stages. Here we describe a zebrafish uox null mutant that possesses elevated urate levels at larval stages. Uricase-deficient 'hyperuricemic' larvae exhibit a suppressed acute inflammatory response to MSU crystals and prolonged in vivo crystal persistence. Imaging of crystals at later stages reveals that they form granuloma-like structures dominated by macrophages. We believe that uox−/− larvae will provide a useful tool to explore the transition from acute gouty inflammation to tophus formation, one of the remaining mysteries of gout pathogenesis. [ABSTRACT FROM AUTHOR]

Hyperuricemia animal models have long been used for evaluating food-derived anti-hyperuricemia compounds. Fructose and potassium oxonate are commonly used for developing hyperuricemia mouse model. Recent research also developed spontaneous hyperuricemia model by uricase knockout (Uox−/−). In this work, we evaluated 3 kinds of models with the same gene background to illustrate the differences between the treatments. Unlike the uric acid levels in potassium oxonate (224.79 ± 33.62 μmol/L) and Uox−/− groups (458.39 ± 38.29 μmol/L), fructose treatment did not lead to higher serum uric acid level (174.93 ± 30.46 μmol/L) comparing to the control group (153.53 ± 40.96 μmol/L). However, abnormal glycometabolism only developed in the fructose and the Uox−/− group. In addition, anemia, inflammasome and severe renal injury occurred in the Uox−/− group. The Uox−/− mice were then treated with puerarin and allopurinol, and found that puerarin could reduce serum uric acid and alleviated the serious renal damage associated with high uric acid. Thus, the Uox−/− mice could be a suitable model for screening and evaluating anti-hyperuricemia compounds. • Three hyperuricemia models were evaluated under the same gene background. • Only Uox-knockout induced abnormal glycometabolism in company with hyperuricemia. • Serious renal damage happened in Uox-knockout mice because of high uric acid level. • Puerarin reduced uric acid level and thus alleviated the consequential renal damage. [ABSTRACT FROM AUTHOR]

Current uric acid detection methodologies lack the requisite sensitivity and selectivity for point-of-care applications. Plasmonic sensors, while promising, demand refinement for improved performance. This work introduces a biofunctionalized sensor predicated on surface plasmon resonance to quantify uric acid within physiologically relevant concentration ranges. The sensor employs the covalent immobilization of uricase enzyme using 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysuccinimide (NHS) crosslinking agents, ensuring the durable adherence of the enzyme onto the sensor probe. Characterization through atomic force microscopy and Fourier transform infrared spectroscopy validate surface alterations. The Langmuir adsorption isotherm model elucidates binding kinetics, revealing a sensor binding affinity of 298.83 (mg/dL)−1, and a maximum adsorption capacity of approximately 1.0751°. The biofunctionalized sensor exhibits a sensitivity of 0.0755°/(mg/dL), a linear correlation coefficient of 0.8313, and a limit of detection of 0.095 mg/dL. Selectivity tests against potentially competing interferents like glucose, ascorbic acid, urea, D-cystine, and creatinine showcase a significant resonance angle shift of 1.1135° for uric acid compared to 0.1853° for interferents at the same concentration. Significantly, at a low uric acid concentration of 0.5 mg/dL, a distinct shift of 0.3706° was observed, setting it apart from the lower values noticed at higher concentrations for all typical interferent samples. The uricase enzyme significantly enhances plasmonic sensors for uric acid detection, showcasing a seamless integration of optical principles and biological recognition elements. These sensors hold promise as vital tools in clinical and point-of-care settings, offering transformative potential in biosensing technologies and the potential to revolutionize healthcare outcomes in biomedicine. [ABSTRACT FROM AUTHOR]

Mini protein mimicking uricase (mp20) has shown significant potential as a replacement for natural enzymes in the development of uric acid biosensors. However, the design of mp20 has resulted to an inactive form of peptide, causing of loss their catalytic activity. Herein, this paper delineates the impact of various metal cofactors on the catalytic activity of mp20. The metal ion-binding site prediction and docking (MIB) web server was employed to identify the metal ion binding sites and their affinities towards mp20 residues. Among the tested metal ions, Cu2+ displayed the highest docking score, indicating its preference for interaction with Thr16 and Asp17 residues of mp20. To assess the catalytic activity of mp20 in the presence of metal ions, uric acid assays was monitored using a colorimetric method. The presence of Cu2+ in the assays promotes the activation of mp20, resulting in a color change based on quinoid production. Furthermore, the encapsulation of the mp20 within zeolitic imidazolate framework-8 (ZIF-8) notably improved the stability of the biomolecule. In comparison to the naked mp20, the encapsulated ZIFs biocomposite (mp20@ZIF-8) demonstrates superior stability, selectivity and sensitivity. ZIF's porous shells provides excellent protection, broad detection (3–100 μM) with a low limit (4.4 μM), and optimal function across pH (3.4–11.4) and temperature (20–100°C) ranges. Cost-effective and stable mp20@ZIF-8 surpasses native uricase, marking a significant biosensor technology breakthrough. This integration of metal cofactor optimization and robust encapsulation sets new standards for biosensing applications. [Display omitted] • A newly designed mini protein mimicking uricase (mp20) served as a bioreceptor for uric acid sensing. • Identification of optimal metal cofactor for mini protein via MIB site prediction and binding server. • ZIF-8 hosts mp20, preserving its catalytic properties with remarkable stability. [ABSTRACT FROM AUTHOR]

Simple Summary: The concentration of uric acid in blood is sex-, age- and diet-dependent and is maintained close to its maximal solubility, indicating that it plays some important role. Indeed, it has been demonstrated that, at physiological concentrations, uric acid is a powerful antioxidant and is a scavenger of singlet oxygen and radicals. At high intracellular concentration, uric acid has been demonstrated to act as a pro-oxidant molecule. Recently, uric acid has been reported to affect the properties of several proteins involved in metabolic regulation and signaling, and the relationship between uric acid and cancer has been extensively investigated. In this review, we present the most recent results on the positive and negative effects played by uric acid in cancer and some new findings and hypotheses about the implication of this metabolite in the pathogenesis of several diseases such as metabolic syndrome, diabetes, and inflammation, thus favoring the development of cancer. Uric acid is the final product of purine catabolism in man and apes. The serum concentration of uric acid is sex-, age- and diet-dependent and is maintained close to its maximal solubility, indicating that it plays some important role. Indeed, it has been demonstrated that, at physiological concentrations, uric acid is a powerful antioxidant, while at high intracellular concentrations, it is a pro-oxidant molecule. In this review, we describe the possible causes of uric acid accumulation or depletion and some of the metabolic and regulatory pathways it may impact. Particular attention has been given to fructose, which, because of the complex correlation between carbohydrate and nucleotide metabolism, causes uric acid accumulation. We also present recent results on the positive and negative effects played by uric acid in cancer and some new findings and hypotheses about the implication of this metabolite in a variety of signaling pathways, which can play a role in the pathogenesis of diseases such as metabolic syndrome, diabetes, and inflammation, thus favoring the development of cancer. The loss of uricase in Homo sapiens and great apes, although exposing these species to the potentially adverse effects of uric acid, appears to be associated with evolutionary advantages. [ABSTRACT FROM AUTHOR]

A label-free electrochemical biosensor has been developed using zinc oxide nanoflowers (ZnONFs) for the detection of uric acid concentration in human blood serum. ZnONFs have been synthesized by a hydrothermal process and characterized with several techniques such as ultraviolet–visible (UV–Vis) spectroscopy, Fourier transform infrared spectroscopy (FTIR) studies, X-ray diffraction (XRD) study, Raman spectroscopy, scanning electron microscopy (SEM) and high-resolution transmission electron microscopy (HR-TEM) and electrochemical analyzer to confirm the formation of nanoflowers and fabrication of electrode and bio-electrodes for uric acid detection. Zinc Oxide nanoflowers have been deposited onto indium–tin oxide (ITO) substrate through electrophoretic deposition technique, and the biosensor has been fabricated by immobilizing urate oxidase (UOx) enzyme onto ZnONFs/ITO electrode surfaces. Further, electrochemical studies have been performed with immobilized UOx/ZnONFs/ITO bio-electrode as a function of uric acid concentrations. It has been found that the fabricated uric acid biosensor shows a high sensitivity (10.38 μA/mM/cm2) and a limit of detection of 0.13 mM in the range of 0.005 to 1.0 mM. This study demonstrates the potential use of ZnONFs for the construction of sensitive biosensors for uric acid detection. [ABSTRACT FROM AUTHOR]

Conventional chemotherapy plays a key role in hepatocellular carcinoma (HCC) treatment, however, with intrinsic or acquired chemoresistance being a major constraint. Here, we aimed to identify potential target to reverse such chemoresistance. In the present study, we found significant difference in uridine monophosphate synthetase (UMPS) expression between 5-FU resistant and sensitive HCC cell lines and the overexpression or downregulation of UMPS impacted 5-FU response in HCC cells. We further found that inhibition of UMPS activity with uric acid at concentration present in human plasma decreased the 5-FU sensitivity of HCC cells, while reduction of uric acid levels with uricase improved the 5-FU sensitivity of HCC cells as well as colorectal cancer cells. In vivo studies also suggested that modulation of uric acid levels did affect 5-FU sensitivity of tumors. These data indicated that UMPS was correlated with the 5-FU resistance in HCC cells and uricase sensitized cancer cells to 5-FU through uricase-uric acid-UMP synthase axis, which provided a potential strategy to improve the efficacy of 5-FU-based chemotherapy for human cancers. [ABSTRACT FROM AUTHOR]

Abstract Background Recent studies suggest that fructose, as well as its metabolite, uric acid, have been associated with increased risk for both cancer incidence and growth. Both substances are known to cause oxidative stress to mitochondria and to reduce adenosine triphosphate (ATP) production by blocking aconitase in the Krebs cycle. The uricase mutation that occurred in the Miocene has been reported to increase serum uric acid and to amplify the effects of fructose to stimulate fat accumulation. Here we tested whether the uricase mutation can also stimulate tumor growth. Methods Experiments were performed in mice in which uricase was inactivated by either knocking out the gene or by inhibiting uricase with oxonic acid. We also studied mice transgenic for uricase. These mice were injected with breast cancer cells and followed for 4 weeks. Results The inhibition or knockout of uricase was associated with a remarkable increase in tumor growth and metastases. In contrast, transgenic uricase mice showed reduced tumor growth. Conclusion A loss of uricase increases the risk for tumor growth. Prior studies have shown that the loss of the mutation facilitated the ability of fructose to increase fat which provided a survival advantage for our ancestors that came close to extinction from starvation in the mid Miocene. Today, however, excessive fructose intake is rampant and increasing our risk not only for obesity and metabolic syndrome, but also cancer. Obesity-associated cancer may be due, in part, to a mutation 15 million years ago that acted as a thrifty gene.

We reported on uric acid (UA) detection using a new fluorescence-based assay: poly-(vinylpyrrolidone) templated copper nanoclusters (PVP-CuNCs) with uricase in an aqueous medium, such as human urine with uricase. These nanoclusters were synthesized in a simple wet chemical method and their morphological and optical properties were examined with the aid of high-resolution transmission electron microscopy and optical absorbance/emission spectroscopy. The PVP-CuNCs acted as the fluorescence indicators that used the enzyme-catalyzed oxidation of UA with uricase. Adding UA into the hybrid PVP-CuNCs/uricase solution caused enzyme-catalyzed oxidation to occur, producing hydrogen peroxide (H2O2), allantoin, and carbon dioxide. The fluorescence intensity of PVP-CuNCs is decreased by this biocatalytically generated H2O2, and this decrease is proportional to the UA level. A calibration plot showed the linear relationship with the negative slope between fluorescence intensity and UA in the range of 5–100 × 10−7 mol/L. The limit of detection (LOD) of UA was estimated as 113 × 10−9 mol/L. This fluorescent probe turned out to be highly specific for UA over other biologically relevant molecules. The demonstrated capability of the PVP-CuNCs as the nanoprobes for quantification of the UA levels in human urine samples could potentially pave the way toward medical applications where a super-sensitive, cost-effective, and UA-specific diagnosis was required.

This work presents the use of encapsulated mini protein 20 mimicking uricase (mp20)-zeolitic imidazolate framework-8 (ZIF-8) as a bioreceptor for the development of a nanozyme-based electrochemical biosensor for uric acid detection. The electrochemical performance of the biofunctionalized mp20@ZIF-8 on the reduced graphene oxide/screen-printed carbon electrode (rGO/SPCE) was investigated by optimizing operating parameters such as pH, deposition potential, and deposition time using a central composite design-response surface methodology (CCD-RSM). The quadratic regression model was developed to correlate the combination of each variable to the oxidation current density as a response. A significant effect on current response was observed under optimized conditions of pH of 7.4 at −0.35 V deposition potential and 56.56 s deposition time, with p < 0.05 for each interacted factor. The obtained coefficient of determination (R2) value of 0.9992 indicated good agreement with the experimental finding. The developed nanozyme biosensor (mp20@ZIF-8/rGO/SPCE) exhibited high selectivity in the presence of the same fold concentration of interfering species with a detection limit of 0.27 μM, over a concentration range of 1 to 34 μM. The practicality of the tailored biosensor in monitoring uric acid in human serum and urine samples was validated with high-performance liquid chromatography (HPLC) and a commercial uric acid meter. Hence, nanozyme-based is a promising platform that offers a rapid, sensitive, selective, and low-cost biosensor for the non-enzymatic detection of uric acid in biological samples. [ABSTRACT FROM AUTHOR]

In this study, a novel nano-biosensor for uric acid detection has been designed and fabricated by embedding uricase into a copper nanozyme, which possessed outstanding peroxidase-like activity and could efficiently mimic horseradish peroxidase (HRP). After optimizing the preparation conditions and characterization, we successfully applied the nano-biosensor to detect uric acid levels. The equation of linear regression was prescribed as y = 0.0064x + 0.3299, while its correlation coefficient is 0.9886. The colorimetric sensor exhibited a linear response range over 1–50 μM and a relatively low limit of detection (LOD) of 0.6 µM. The nano-biosensor has the advantages of easy preparation, good reusability and excellent storage stability, while this one-step colorimetric method is simple to be operated and has high selectivity for detection of the uric acid levels in actual human samples. [ABSTRACT FROM AUTHOR]

A flow-through biosensor has been developed and manufactured by 3D printing from poly(lactic acid). Uricase or tyrosinase were immobilized on the inner walls of the reactor cell and the concentrations of the reaction products, i.e., hydrogen peroxide in the case of uricase and quinone product of the tyrosine oxidation in the case of tyrosinase, were monitored in the amperometric mode. The screen-printed electrode modified with carbon black, pillar[5]arene and electropolymerized thionine and methylene blue was separated from the enzyme layer by 0.1 mm gap and used as a biosensor transducer. The components of the modifier layer exerted a synergetic effect and increased the cathodic current up to seven times compared to electrodes modified with individual components, increasing the sensitivity of the analysis. In the optimal conditions, biosensor allowed the determination of 1 × 10−7 to 1 × 10−5 M uric acid and 2 × 10−7 to 1 × 10−5 M tyrosine. The biosensors analyzed spiked samples of blood serum and urine and provided recoveries from 95 to 110%. Easy mounting of the flow-through cell and low cost of the replaceable parts make promising future application of the biosensor for routine clinical assays. [ABSTRACT FROM AUTHOR]

The development of multifunctional and high-performance nanozymes via a facile strategy is especially intriguing for biomedical applications. Here, a surfactant-free simple method was used to successfully load small-sized Pt nanoflowers (PtNFs) onto reduced graphene oxide (rGO) based on single glucose. PtNF@rGO nanozyme (NM) exhibits highly excellent catalytic activity, and displays good stability and recyclability in degrading uric acid. More importantly, this robust integrated nanozyme shows strong sulfur resistance. The analysis of the structure-function relationship indicates that the small sized nanoflower structure and the interaction between PtNF and rGO in the integrated catalysts contribute to the catalytic activity and stability collectively. Interestingly, PtNF@rGO nanozymes can mimic the cascade processes of uricase/catalase to degrade both uric acid and H 2 O 2. Furthermore, the inhibition effect of PtNF@rGO on the crystallization of urate crystals was investigated. PtNF@rGO nanozyme prolongs the nucleation induction time (from 10 to 72 h) during the crystallization of sodium urate significantly. This study reveals the significance of morphology regulation in constructing high-performance and multifunctional hybrid nanozymes, broadening the application of nanozyme in the biomedical field. [Display omitted] • Small-size Pt nanoflower (PtNF)@rGO hybrid is facilely prepared based on single glucose. • Synergistic effects of PtNF and rGO enhance uricase-like activity of the hybrid highly. • PtNF@rGO hybrid nanozyme shows strong sulfur resistance. • Self-cascade uricase/peroxidase mimics is obtained based on PtNF@rGO hybrid. • PtNF@rGO hybrid inhibits the crystallization of sodium urate significantly. [ABSTRACT FROM AUTHOR]

In recent years, studies on the external stimulation of biotechnological enzyme drugs and their use in the treatment of diseases have gradually increased. Herein, for the first time, the near-infrared (NIR) was used as an external stimulant to manipulate the catalytic activity of the uricase (UOx) enzyme, which is used in the treatment of hyperuricemia, in a controlled manner. For this purpose, NaYF 4 : Yb3+, Er3+ upconverting nanoparticles (UCNPs) were synthesized by hydrothermal synthesis method and functionalized with diethylaminoethyldextran (DEAE-D) to facilitate UOx immobilization. The obtained materials were characterized in detail by various methods to confirm the preparation of UCNPs and immobilization of UOx. In addition, the biochemical parameters such as optimum pH, optimum temperature, thermal stability, and reusability were preliminarily investigated for free UOx and NaYF 4 : Yb3+, Er3+/DEAE-D/UOx. Moreover, a sequential experimental method was monitored to assess the effects of NIR excitation intensity, induction distance, and exposure time on the UOx activity. While the optimum pH value was found to be 6.0 for both enzyme forms, the optimum temperature value was recorded as 45 and 50 °C for free UOx and NaYF 4 : Yb3+, Er3+/DEAE-D/UOx, respectively. The activation energy (E a) values of free UOx and NaYF 4 : Yb3+, Er3+/DEAE-D/UOx were calculated to be 7.59 and 2.98 kJ/mol, respectively, implying that the NaYF 4 : Yb3+, Er3+/DEAE-D/UOx was less temperature sensitive. After thermal incubation for 3 h at 55 °C, the NaYF 4 : Yb3+, Er3+/DEAE-D/UOx retained 54.68 % of its initial activity, while the free UOx retained 32.94 % of its initial activity at 50 °C. In addition, the findings from the reusability experiments revealed that NaYF 4 :Yb3+, Er3+/DEAE-D/UOx retained 57.94 % of its initial activity even after 10 reuse cycles. The most striking point in this study was the positive manipulation of UOx activity by NIR. Accordingly, it was observed that when the NIR power was 1500 mW, the UOx activity increased about 2 times compared to the control. Additionally, the UOx activity increased in parallel with the increase in NIR application time and the ideal application distance was 3 cm. In conclusion, this pioneering study provides valuable insights into the controlled manipulation of enzyme activity, showcasing the effectiveness of NIR in enhancing enzyme activity. The outcomes suggest that NIR holds great promise as an efficient, sustainable, and versatile approach applicable to various enzymatic catalysis scenarios. • A novel support (NaYF 4 : Yb3+, Er3+/DEAE-D) was synthesized for UOx immobilization. • Physicochemical and biochemical characterizations were carried out. • NaYF 4 : Yb3+, Er3+/DEAE-D/UOx had hydrodynamic diameter of 415.08 nm and zeta potential of 0.99 mV. • NaYF 4 : Yb3+, Er3+/DEAE-D/UOx showed relative activity of 57.94 % after 10 cycles. • More than 2-fold activity was achieved with immobilized UOx under NIR irradiation. [ABSTRACT FROM AUTHOR]

Protein therapeutics, except for antibodies, have a short plasma half‐life and poor stability in circulation. Covalent coupling of polyethylene glycol (PEG) to protein drugs addresses this limitation. However, unlike previously thought, PEG is immunogenic. In addition to induced PEG antibodies, ≈70% of the US population has pre‐existing anti‐PEG antibodies. Both induced and preexisting anti‐PEG antibodies result in accelerated drug clearance, reduced clinical efficacy, and severe hypersensitivity reactions that have limited the clinical utility of uricase, an enzyme drug for treatment for refractory gout that is decorated with a PEG corona. Here, the authors synthesize a poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) conjugate of uricase that decorates the protein with multiple polymer chains to create a corona to solve these problems. The resulting uricase‐POEGMA is well‐defined, has high bioactivity, and outperforms its PEG counterparts in its pharmacokinetics (PK). Furthermore, the conjugate does not induce anti‐POEGMA antibodies and is not recognized by anti‐PEG antibodies. These findings suggest that POEGMA conjugation may provide a solution to the immunogenicity and antigenicity limitations of PEG while improving upon its PK benefits. These results transcend uricase and can be applied to other PEGylated therapeutics and the broader class of biologics with suboptimal PK. [ABSTRACT FROM AUTHOR]