Search Results (20)

Enzymatic modification of Kraft lignin under alkaline conditions was investigated using bilirubin oxidase (BOD) in borate buffer (pH 10). Control solubilization without enzyme addition revealed a notable increase in molar mass (up to 1.7-fold) and potential borate complexation with lignin hydroxyl groups, as evidenced by thermogravimetric and ¹¹B NMR analyses. BOD treatments induced substantial polymerization, with molar mass increases of up to 4-fold for insoluble fractions after 24 h, while soluble fractions exhibited progressive increases over 5 days. Quantitative ³¹P NMR showed reductions in aliphatic and phenolic hydroxyl groups by 20%, suggesting oxidative coupling reactions, particularly through 4-O-5′ and 5-5′ linkages. Solid-state ¹³C NMR confirmed structural changes associated with polymerization. Dynamic light scattering (DLS) indicated the presence of colloidal aggregates, potentially explaining challenges in HSQC NMR signal acquisition. These findings highlight the efficacy of bilirubin oxidase in catalyzing lignin polymerization and underscore the structural impact of borate–lignin interactions in alkaline media, paving the way for advanced lignin valorization strategies. Full article

Bilirubin (BR) is an important ingredient of a valuable Chinese medicine, Calculus bovis. Over recent decades, increasing evidence has confirmed that BR offers health benefits in cardiovascular health, stroke, diabetes, and metabolic syndrome. However, BR is mainly produced by extraction from pig bile. In this study, we assembled an efficient pathway for BR production by metabolic engineering of Escherichia coli. First, heme oxygenase (HO1) and biliverdin reductase were co-expressed in E. coli. HPLC and LC–MS confirmed the accumulation of BR in the recombinant E. coli cells. To improve BR production, the catalytic abilities of HO1 from different species were investigated. In addition, the outermembrane-bound heme receptor (ChuA) and the enzymes involved in heme biosynthesis were overexpressed among which ChuA, 5-aminolevulinic acid dehydratase (HemB), protoporphyrin oxidase (HemG), and ferrochelatase (HemH) were found to enhance BR accumulation in E. coli. In addition, expression of ferredoxin (Fd) was shown to contribute to efficient conversion of heme to BR in E. coli. To increase supply of NADPH, isocitrate dehydrogenase (IDH), NAD kinase (nadK), NADP-specific glutamate dehydrogenase (gdhA), and glucose-6-phosphate 1-dehydrogenase (ZWF) were overexpressed and were found to enhance BR accumulation when these proteins were expressed with a low-copy plasmid pACYCduet-1. Modular optimization of the committed genes led to a titer of 17.2 mg/L in strain M1BHG. Finally, fed-batch fermentation was performed for the strains M1BHG and M1, resulting in accumulation of 75.5 mg/L and 25.8 mg/L of BR, respectively. This is the first report on biosynthesis of BR through metabolic engineering in a heterologous host. Full article

This study aimed to comprehensively assess the metabolic, mitochondrial, and inflammatory effects of first-line efavirenz, emtricitabine, and tenofovir disoproxil fumarate (EFV/FTC/TDF) single-tablet regimen (STR) relative to untreated asymptomatic HIV infection. To this end, we analyzed 29 people with HIV (PWH) treated for at least one year with this regimen vs. 33 antiretroviral-naïve PWH. Excellent therapeutic activity was accompanied by significant alterations in metabolic parameters. The treatment group showed increased plasmatic levels of glucose, total cholesterol and its fractions (LDL and HDL), triglycerides, and hepatic enzymes (GGT, ALP); conversely, bilirubin levels (total and indirect fraction) decreased in the treated cohort. Mitochondrial performance was preserved overall and treatment administration even promoted the recovery of mitochondrial DNA (mtDNA) content depleted by the virus, although this was not accompanied by the recovery in some of their encoded proteins (since cytochrome c oxidase II was significantly decreased). Inflammatory profile (TNFα, IL-6), ameliorated after treatment in accordance with viral reduction and the recovery of TNFα levels correlated to mtDNA cell restoration. Thus, although this regimen causes subclinical metabolic alterations, its antiviral and anti-inflammatory properties may be associated with partial improvement in mitochondrial function. Full article

Ordered mesoporous carbon CMK-3 sieves with a hexagonal structure and uniform pore size have recently emerged as promising materials for applications as adsorbents and electrodes. In this study, using sucrose as the sustainable carbon source and SBA-15 as a template, CMK-3 sieves are synthesized to form bioelectrocatalytic immobilization matrices for enzymatic biofuel cell (EFC) electrodes. Their electrochemical performance, capacitive features, and the stability of enzyme immobilization are analyzed and compared to commercially available multi-walled carbon nanotubes (MWCNT) using cyclic voltammetry and electrochemical impedance spectroscopy (EIS). The anodic reaction in the presence of glucose oxidase (GOx) and ferrocene methanol (FcMeOH) on the sustainably sourced CMK-3-based electrodes produces bioelectrocatalytic current responses at 0.5 V vs. saturated calomel electrode (SCE) that are twice as high as on the MWCNT-based electrodes under saturated glucose conditions. For the cathodic reaction, the MWCNT-based cathode performs marginally better than the CMK-3-based electrodes in the presence of bilirubin oxidase (BOD) and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS²⁻). The CMK-3-based EFCs assembled from the GOx anode and BOD cathode results in a power output of 93 μW cm⁻². In contrast, the output power of MWCNT-based EFCs is approximately 53 μW cm⁻². The efficiency of CMK-3 as a support material for biofuel cell applications is effectively demonstrated. Full article

Biofuel cells (BFCs) with enzymatic electrocatalysts have attracted significant attention, especially as power sources for wearable and implantable devices; however, the applications of BFCs are limited owing to the limited O₂ supply. This can be addressed by using air-diffusion-type bilirubin oxidase (BOD) cathodes, and thus the further development of the hierarchical structure of porous electrodes with highly effective specific surface areas is critical. In this study, a porous layer of gold is deposited over magnesium-oxide-templated carbon (MgOC) to form BOD-based biocathodes for the oxygen reduction reaction (ORR). Porous gold structures are constructed via electrochemical deposition of gold via dynamic hydrogen bubble templating (DHBT). Hydrogen bubbles used as a template and controlled by the Coulomb number yield a porous gold structure during the electrochemical deposition process. The current density of the ORR catalyzed by BOD without a redox mediator on the gold-modified MgOC electrode was 1.3 times higher than that of the ORR on the MgOC electrode. Furthermore, the gold-deposited electrodes were modified with aromatic thiols containing negatively charged functional groups to improve the orientation of BOD on the electrode surface to facilitate efficient electron transfer at the heterogeneous surface, thereby achieving an ORR current of 12 mA cm⁻² at pH 5 and 25 °C. These results suggest that DHBT is an efficient method for the fabrication of nanostructured electrodes that promote direct electron transfer with oxidoreductase enzymes. Full article

Brussels chicory, a typical vegetable in Mediterranean diets, has been recently reported to stabilize advanced atherosclerotic plaques in the brachiocephalic artery of apoE-deficient (Apoe^−/−) mice. Herein, we investigated whether Brussels chicory can stabilize advanced plaques in the aorta via improving oxidative stress. Thirty week old Apoe^−/− mice were fed the AIN-93G diet or supplemented with 0.5% freeze-dried Brussels chicory for twenty weeks. Aortic plaque size and stability, aortic relaxation, monocyte adhesion to aortic endothelium, free radicals, and enzymatic and non-enzymatic factors involved in free radical production and elimination in aorta and serum were measured. Brussels chicory consumption did not alter aortic plaque size, however, it stabilized aortic plaques, promoted aortic relaxation, and also inhibited monocyte adhesion to aortic endothelium. Moreover, this administration reduced oxidized LDL (ox-LDL) and 4-hydroxynonenal (4-HNE) content in aortic plaques, associated with inhibited aortic NADPH oxidase (NOX) and uncoupled endothelial nitric oxide synthase (eNOS)-mediated free radical production. However, Brussels chicory consumption did not appreciably alter aortic and serum superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, aortic glutathione (GSH), as well as serum non-enzymatic antioxidants, such as bilirubin, uric acid, and GSH. Collectively, improved oxidative stress might contribute to the atheroprotective effect of Brussels chicory, supporting the prospect of the antioxidant therapy in advanced atherosclerosis progression. Full article

Herein, a paper-based glucose/air biofuel cell (BFC) was constructed and implemented for self-powered pesticide detection. Our developed paper-based chip relies on a hollow-channel to transport fluids rather than capillarity, which reduces analysis times as well as physical absorption. The gold nanoparticles (Au NPs) and carbon nanotubes (CNTs) were adapted to modify the paper fibers to fabricate the flexible conductive paper anode/cathode electrode (Au–PAE/CNT–PCE). Molecularly imprinted polymers (MIPs) using 2,4-dichlorophenoxyacetic acid (2,4-D) as a template were synthesized on Au–PAE for signal control. In the cathode, bilirubin oxidase (BOD) was used for the oxygen reduction reaction. Based on a competitive reaction between 2,4-D and glucose-oxidase-labeled 2,4-D (GOx-2,4-D), the amount of GOx immobilized on the bioanode can be simply tailored, thus a signal-off self-powered sensing platform was achieved for 2,4-D determination. Meanwhile, the coupling of the paper supercapacitor (PS) with the paper-based chip provides a simple route for signal amplification. Combined with a portable digital multi-meter detector, the amplified signal can be sensitively readout. Through rational design of the paper analytical device, the combination of BFC and PS provides a new prototype for constructing a low-cost, simple, portable, and sensitive self-powered biosensor lab-on-paper, which could be easily expanded in the field of clinical analysis and drug delivery. Full article

Hyperuricemia is the chief cause of gout and has been linked with hypertension, cardiovascular and renal disease, diabetes and metabolic syndrome. Liver with the highest protein expression of xanthine oxidase, the main enzyme responsible for uric acid formation, is the primary site of uric acid biosynthesis. However, there are few studies that examine the association between liver function and new-onset hyperuricemia. Hence, using the Taiwan Biobank dataset, we aimed to explore the capability of liver function parameters, including gamma-glutamyl transferase, total bilirubin, albumin, alanine aminotransferase and aspartate aminotransferase in association with the subsequent development of hyperuricemia. We analyzed 21,030 participants without hyperuricemia at baseline. Hyperuricemia was defined as a uric acid concentration > 6.0 mg/dL in women or >7.0 mg/dL in men. New-onset hyperuricemia was defined as participants without baseline hyperuricemia having developed hyperuricemia upon subsequent exam. Overall, 1804 (8.6%) of the study subjects developed new-onset hyperuricemia. After multivariable analysis, significant associations were found between the male sex (odds ratio [OR], 4.412; p < 0.001), high values of systolic blood pressure (SBP) (OR, 1.006; p = 0.012), body mass index (BMI) (OR, 1.064; p < 0.001), fasting glucose (OR, 1.005; p < 0.001), triglycerides (OR, 1.001; p = 0.003), uric acid (OR, 5.120; p < 0.001), low values of estimated glomerular filtration rates (eGFR) (OR, 0.995; p < 0.001), total bilirubin (OR, 0.616; p < 0.001) and new-onset hyperuricemia. The cutoff level of total bilirubin, according to the Youden index, of receiver operating characteristic curve for identifying new-onset hyperuricemia was 0.65 mg/dL. Low total bilirubin was defined as ≤0.65 mg/dL. After multivariable analysis, we found a significant association between low total bilirubin level (≤0.65 mg/dL) (OR = 0.806; p < 0.001) and new-onset hyperuricemia. Our present study demonstrated that in addition to male sex, high SBP, BMI, fasting glucose, triglycerides, and uric acid and low eGFR, the serum’s total bilirubin levels were negatively associated with new-onset hyperuricemia in a large Taiwanese cohort. Full article

A notable diagnostic for the detection of hemolytic diseases is bilirubin, a by-product of haemoglobin breakdown. The concentration of bilirubin ranges from 0.3 to 1.9 mg in 100 mL of blood. Low blood bilirubin levels are associated with a greater risk of coronary heart disease and anaemia. Hyperbilirubinemia results from a serum bilirubin level of more than 2.5 mg/100 mL. Therefore, it is very crucial to check the serum bilirubin level. Analytical equipment for point-of-care testing must be portable, small, and affordable. A unique method is used to detect bilirubin selectively using paper-based screen-printed carbon electrodes that were covalently linked with nanoparticles, that serves as a key biomarker for jaundice. In order to create an electrochemical biosensor, bilirubin oxidase was immobilised on electrodes modified with AgNPs. The morphology of Ag nanoparticles in terms of size and shape was determined using both UV- Vis Spectroscopy and transmission electron microscopy (TEM). The biosensor’s analytical response was assessed using potentiostat (Cyclic voltammetry (CV) and linear sweep voltammetry (LSV)). The developed paper-based sensor provided optimum feedback and a broad linear range of 1 to 9 µg/mL for bilirubin, with a lower LOD of 1 µg/mL. Through tests of bilirubin in artificial blood serum, the viability is confirmed. The method that is being used makes it possible to create and use an inexpensive, miniature electrochemical sensor. Full article

A new concept of hollow electrode based on the assembly of two buckypapers creating a microcavity which contains a biocatalyst is described. To illustrate this innovative concept, hollow bioelectrodes containing 0.16–4 mg bilirubin oxidase in a microcavity were fabricated and applied to electroenzymatic reduction of O₂ in aqueous solution. For hemin-modified buckypaper, the bioelectrode shows a direct electron transfer between multi-walled carbon nanotubes and bilirubin oxidase with an onset potential of 0.77 V vs. RHE. The hollow bioelectrodes showed good storage stability in solution with an electroenzymatic activity of 30 and 11% of its initial activity after 3 and 6 months, respectively. The co-entrapment of bilirubin oxidase and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) in the microcavity leads to a bioelectrode exhibiting mediated electron transfer. After 23 h of intermittent operation, 5.66 × 10⁻⁴ mol of O₂ were electroreduced (turnover number of 19,245), the loss of catalytic current being only 54% after 7 days. Full article

Efficient oxygen-reducing biocatalysts are essential for the development of biofuel cells or photo-bioelectrochemical applications. Bilirubin oxidase (BOD) is a promising biocatalyst for oxygen reduction processes at neutral pH and low overpotentials. BOD has been extensively investigated over the last few decades. While the enzyme’s internal electron transfer process and methods to establish electrical communication with electrodes have been elucidated, a crystal structure of BOD from bacterial origin has never been determined. Here we present the first crystal structure of BOD from Bacillus pumilus (BpBOD) at 3.5 Å resolution. Overall, BpBOD shows high homology with the fungal enzymes; however, it holds a unique surface-exposed disulfide bond between Cys229 and Cys322 residues. We present methodologies to orient the T1 site towards the electrode by coupling the reduced disulfide bond with maleimide moiety on the electrodes. The developed configurations were further investigated and revealed improved direct electron transfer rates with the electrodes. The work presented here may contribute to the construction of rationally designed bioanodes or biocathode configurations that are based on redox-active enzymes. Full article

Bilirubin has been regarded as a powerful endogenous antioxidant and anti-inflammatory molecule, able to act on cellular pathways as a hormone. Diabetic kidney disease (DKD) is a common chronic complication of diabetes, and it is the leading cause of end-stage renal disease. Here, we will review the clinical and molecular features of mild hyperbilirubinemia in DKD. The pathogenesis of DKD involves oxidative stress, inflammation, fibrosis, and apoptosis. Serum bilirubin levels are positively correlated with the levels of the antioxidative enzymes as superoxide dismutase, catalase, and glutathione peroxidase, while it is inversely correlated with C-reactive protein, TNF-α, interleukin (IL)-2, IL-6, and IL-10 release in diabetic kidney disease. Bilirubin downregulates NADPH oxidase, reduces the induction of pro-fibrotic factor HIF-1α expression, cleaved caspase-3, and cleaved PARP induction showing lower DNA fragmentation. Recent experimental and clinical studies have demonstrated its effects in the development and progression of renal diseases, pointing out that only very mild elevations of bilirubin concentrations result in real clinical benefits. Future controlled studies are needed to explore the precise role of bilirubin in the pathogenesis of DKD and to understand if the use of serum bilirubin levels as a marker of progression or therapeutic target in DKD is feasible and realistic. Full article

Liver failure is associated with increased levels of brain aromatic amino acids (AAAs), whose transport across the blood–brain barrier (BBB) is mainly mediated by L-amino acid transporter 1 (LAT1). We aimed to investigate whether liver failure induced by bile duct ligation (BDL) increases levels of brain AAAs by affecting the expression and function of LAT1. The LAT1 function was assessed using the brain distribution of gabapentin. It was found that BDL significantly increased levels of gabapentin, phenylalanine, and tryptophan in the cortex, hippocampus, and striatum of rats, and upregulated the expression of total LAT1 protein in hippocampus and striatum as well as cortex membrane LAT1 protein. HCMEC/D3 served as in vitro BBB model, and the data showed that both the serum of BDL rats and bilirubin induced LAT1 expression and function, while bilirubin oxidase almost abolished the upregulation of LAT1 protein by bilirubin and the serum of BDL rats. The enhanced function and expression of LAT1 were also observed in the hippocampus and striatum of hyperbilirubinemia rats. Both aryl hydrocarbon receptor (AhR) antagonist α-naphthoflavone and AhR silencing obviously attenuated the upregulation of LAT1 protein by bilirubin or omeprazole. This study provides the first evidence that BDL upregulates LAT1 at the rat BBB, attributed to the activation of AhR by the increased plasma bilirubin. The results highlight the mechanisms causing BDL-increased levels of brain AAAs and their physiological significance. Full article

The glucose oxidase–peroxidase (GOD–POD) method used to measure serum unbound bilirubin (UB) suffers from direct bilirubin (DB) interference. Using a bilirubin-inducible fluorescent protein from eel muscle (UnaG), a novel GOD–POD–UnaG method for measuring UB was developed. Newborn sera with an indirect bilirubin/albumin (iDB/A) molar ratio of <0.5 were classified into four groups of DB/total serum bilirubin (TB) ratios (<5%, 5–10%, 10–20%, and ≥20%), and the correlation between the UB levels and iDB/A ratio was examined. Linear regression analysis was performed to compare UB values from both methods with the iDB/A ratio from 38 sera samples with DB/TB ratio <5% and 11 samples with DB/TB ratio ≥5%. The correlation coefficient (r) between UB values and the iDB/A ratio for the GOD–POD method was 0.8096 (DB/TB ratio <5%, n = 239), 0.7265 (5–10%, n = 29), 0.7165 (10–20%, n = 17), and 0.4816 (≥20%, n = 16). UB values using the GOD–POD–UnaG method highly correlated with the iDB/A ratio in both <5% and ≥5% DB/TB ratio sera (r = 0.887 and 0.806, respectively), whereas a low correlation (r = 0.428) occurred for ≥5% DB/TB ratio sera using the GOD–POD method. Our GOD–POD–UnaG method can measure UB levels regardless of the presence of DB. Full article

Glutathione is an important biological molecule which can be an indicator of numerous diseases. A method for self-powered detection of glutathione levels in solution has been developed using an enzymatic biofuel cell. The device consists of a glucose oxidase anode and a bilirubin oxidase cathode. For the detection of glutathione, the inhibition of bilirubin oxidase leads to a measurable decrease in current and power output. The reported method has a detection limit of 0.043 mM and a linear range up to 1.7 mM. Being able to detect a range of concentrations can be useful in evaluating a patient’s health. This method has the potential to be implemented as a quick, low-cost alternative to previously reported methods. Full article