Glycolysis and de novo fatty acid synthesis cooperatively regulate pathological vascular smooth muscle cell phenotypic switching and neointimal hyperplasia.

Switching of vascular smooth muscle cells (VSMCs) from a contractile phenotype to a dedifferentiated (proliferative) phenotype contributes to neointima formation, which has been demonstrated to possess a tumor-like nature. Dysregulated glucose and lipid metabolism is recognized as a hallmark of tumors but has not thoroughly been elucidated in neointima formation. Here, we investigated the cooperative role of glycolysis and fatty acid synthesis in vascular injury-induced VSMC dedifferentiation and neointima formation. We found that the expression of hypoxia-inducible factor-1α (HIF-1α) and its target 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), a critical glycolytic enzyme, were induced in the neointimal VSMCs of human stenotic carotid arteries and wire-injured mouse carotid arteries. HIF-1α overexpression led to elevated glycolysis and resulted in a decreased contractile phenotype while promoting VSMC proliferation and activation of the mechanistic target of rapamycin complex 1 (mTORC1). Conversely, silencing Pfkfb3 had the opposite effects. Mechanistic studies demonstrated that glycolysis generates acetyl coenzyme A to fuel de novo fatty acid synthesis and mTORC1 activation. Whole-transcriptome sequencing analysis confirmed the increased expression of PFKFB3 and fatty acid synthetase (FASN) in dedifferentiated VSMCs. More importantly, FASN upregulation was observed in neointimal VSMCs of human stenotic carotid arteries. Finally, interfering with PFKFB3 or FASN suppressed vascular injury-induced mTORC1 activation, VSMC dedifferentiation, and neointima formation. Together, this study demonstrated that PFKFB3-mediated glycolytic reprogramming and FASN-mediated lipid metabolic reprogramming are distinctive features of VSMC phenotypic switching and could be potential therapeutic targets for treating vascular diseases with neointima formation. © 2023 The Pathological Society of Great Britain and Ireland.

Glycolysis inhibition ameliorates brain injury after ischemic stroke by promoting the function of myeloid-derived suppressor cells.

Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells which are immunosuppressive and glycolytically inactive in inflammatory diseases. However, it is unknown whether MDSCs contribute to ischemic stroke and how glycolysis regulates MDSC function in such a context. Here, we showed that MDSCs arise in the blood of patients at early phase of stroke. Similar results were observed in temporary middle cerebral artery occlusion-induced cerebral ischemic mice. Pharmaceutical exhaustion of MDSCs aggravated, while adoptive transfer of MDSCs rescued the ischemic brain injury. However, the differentiation of MDSCs into immunopotent myeloid cells which coincides with increased glycolysis was observed in the context of ischemic stroke. Mechanistically, the glycolytic product lactate autonomously induces MDSC differentiation through activation of mTORC1, and paracrinely activates Th1 and Th17 cells. Moreover, gene knockout or inhibition of the glycolytic enzyme PFKFB3 increased endogenous MDSCs by blocking their differentiation, and improved ischemic brain injury. Collectively, these results revealed that glycolytic switch decreases the immunosuppressive and neuroprotective role of MDSCs in ischemic stroke and pharmacological targeting MDSCs via glycolysis inhibition constitutes a promising therapeutic strategy for ischemic stroke.

Endothelial nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome regulation in atherosclerosis.

AIMS: The activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in endothelial cells (ECs) contributes to vascular inflammation in atherosclerosis. Considering the high glycolytic rate of ECs, we delineated whether and how glycolysis determines endothelial NLRP3 inflammasome activation in atherosclerosis. METHODS AND RESULTS: Our results demonstrated a significant up-regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key regulator of glycolysis, in human and mouse atherosclerotic endothelium, which positively correlated with NLRP3 levels. Atherosclerotic stimuli up-regulated endothelial PFKFB3 expression via sterol regulatory element-binding protein 2 (SREBP2) transactivation. EC-selective haplodeficiency of Pfkfb3 in Apoe-/- mice resulted in reduced endothelial NLRP3 inflammasome activation and attenuation of atherogenesis. Mechanistic investigations revealed that PFKFB3-driven glycolysis increased the NADH content and induced oligomerization of C-terminal binding protein 1 (CtBP1), an NADH-sensitive transcriptional co-repressor. The monomer form, but not the oligomer form, of CtBP1 was found to associate with the transcriptional repressor Forkhead box P1 (FOXP1) and acted as a transrepressor of inflammasome components, including NLRP3, caspase-1, and interleukin-1ß (IL-1ß). Interfering with NADH-induced CtBP1 oligomerization restored its binding to FOXP1 and inhibited the glycolysis-dependent up-regulation of NLRP3, Caspase-1, and IL-1ß. Additionally, EC-specific overexpression of NADH-insensitive CtBP1 alleviates atherosclerosis. CONCLUSION: Our findings highlight the existence of a glycolysis-dependent NADH/CtBP/FOXP1-transrepression pathway that regulates endothelial NLRP3 inflammasome activation in atherogenesis. This pathway represents a potential target for selective PFKFB3 inhibitors or strategies aimed at disrupting CtBP1 oligomerization to modulate atherosclerosis.

Simultaneous analysis of dopamine and homovanillic acid by high-performance liquid chromatography with wall-jet/thin-layer electrochemical detection.

Wall-jet/thin-layer amperometric electrochemical detection (ECD) coupled with high-performance liquid chromatography (HPLC) was used here for the simultaneous analysis of dopamine (DA) and homovanillic acid (HVA) at a glassy carbon electrode. Compared with the conventional thin-layer mode and wall-jet mode, the presented wall-jet/thin-layer ECD has the advantages of enhanced capture of electroactive DA and HVA on the working electrode to give enhanced responses and more convenient washing/refreshment of the working electrode surface. Under optimized conditions, the HPLC-ECD calibration curves show good linearity from 0.01 to 100 µM for DA and HVA, and the limits of detection (LODs) obtained were 1.1 nM for DA and 0.7 nM for HVA which are lower than those obtained with an UV-vis detector and a commercial electrochemical detector. The method was tested on human urine with satisfactory results. The balance of response-signal, signal-background and noise level for our HPLC-ECD system is also discussed. In addition, a demethylation electrooxidation mechanism for HVA is suggested through potentiostatic bulk electrolysis, electrospray ionization-mass spectrometry, fluorescent spectrophotometry and cyclic voltammetry studies.

High-performance sorbents from ionic liquid activated walnut shell carbon: an investigation of adsorption and regeneration.

In this paper, walnut shells were selected to make activated charcoal using ionic activators. Based on the physical/chemical activation process and the properties of activated carbon products, the Fourier Transform Infrared reflection and Brunauer-Emmett-Teller analysis methods were adopted to comparatively analyse activation principles and pore-structure parameters. Also ciprofloxacin adsorption was compared among various activated carbon. Then, an absence of microporous structure in both walnut shells and their carbonized derivatives was found. Moreover, the specific surface area of activated carbon, prepared via KOH wet activation within physical/chemical procedures, attains a noteworthy 1787.06 m2 g-1, underlining its commendable adsorption performance. The specific surface areas of five distinct activated carbons, processed via ionic activation, extend from 1302.01 to 2214.06 m2 g-1. Concurrently, the micropore volumes span from 0.47 to 0.93 cm3 g-1. Obviously, the adsorption proficiency of ion-activated carbon markedly exceeds that of carbons activated physically or chemically. Of all materials investigated in this paper, ion-activated carbon D consistently exhibits superior performance, maintaining a ciprofloxacin removal rate nearing 100% at 40 °C. Remarkably, the maximum regeneration frequency of ion-activated carbons can reach up to 10 cycles. In conclusion, these five ion-activated carbons, demonstrating superior pore-structure parameters and adsorptive capacities, outperform those prepared through physical/chemical methods. They emerge as promising contenders for new, high-performing adsorbents.

Glycolysis Promotes Angiotensin II-Induced Aortic Remodeling Through Regulating Endothelial-to-Mesenchymal Transition via the Corepressor C-Terminal Binding Protein 1.

BACKGROUND: Endothelial dysfunction plays a crucial role in aortic remodeling. Aerobic glycolysis and endothelial-to-mesenchymal transition (EndoMT) have, respectively, been suggested to contribute to endothelial dysfunction in many cardiovascular diseases. Here, we tested the hypothesis that glycolytic reprogramming is critical for EndoMT induction in aortic remodeling through an epigenetic mechanism mediated by a transcriptional corepressor CtBP1 (C-terminal binding protein 1), a sensor of glycolysis-derived NADH. METHODS: EndoMT program, aortic remodeling, and endothelial expression of the glycolytic activator PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3) were evaluated in Ang (angiotensin) II-infused mice. Mice with endothelial-specific Pfkfb3 deficiency or CtBP1 inactivation, immunoprecipitation, chromatin immunoprecipitation, and luciferase reporter assay were employed to elucidate whether and how PFKFB3/CtBP1 epigenetically controls EndoMT. RESULTS: The EndoMT program and increased endothelial PFKFB3 expression were induced in remodeled thoracic aortas. In TGF-ß (transforming growth factor-ß)-treated human endothelial cells, activated SMAD2/3 (SMAD Family Member 2/3) transcriptionally upregulated PFKFB3 expression. In turn, the TGF-ß/SMAD signaling and EndoMT were compromised by silencing or inhibition of PFKFB3. Mechanistic studies revealed that PFKFB3-mediated glycolysis increased NADH content and activated the NADH-sensitive CtBP1. Through interaction with the transcription repressor E2F4 (E2F Transcription Factor 4), CtBP1 enhanced E2F4-mediated transcriptional repression of SMURF2 (SMAD ubiquitin regulatory factor 2), a negative regulator of TGF-ß/SMAD2 signaling. Additionally, EC-specific Pfkfb3 deficiency or CtBP1 inactivation in mice led to attenuated Ang II-induced aortic remodeling. CONCLUSIONS: Our results demonstrate a glycolysis-mediated positive feedback loop of the TGF-ß signaling to induce EndoMT and indicate that therapeutically targeting endothelial PFKFB3 or CtBP1 activity could provide a basis for treating EndoMT-linked aortic remodeling.

Gene-dosage effect of Pfkfb3 on monocyte/macrophage biology in atherosclerosis.

BACKGROUND AND PURPOSE: Macrophage-rich atherosclerotic arteries are highly active in glycolysis. PFKFB3, a key glycolytic enzyme, has emerged as a potential therapeutic target in atherosclerosis. Small-molecule inhibitors of PFKFB3, such as 3PO and PFK158, have demonstrated efficacy in hampering atherogenesis in preclinical models. However, genetic studies elucidating the role of Pfkfb3 in atherogenesis need to be conducted to validate pharmacological findings and to unveil potential pharmacological side effects. EXPERIMENTAL APPROACH: Apoe-/- mice with global heterozygous or myeloid cell-specific Pfkfb3 deficiency were fed a Western diet (WD), after which atherosclerosis development was determined. Monocyte subsets in atherosclerotic mice and patients were examined by flow cytometry. Monocyte infiltration was assayed by a Ly6Chi monocyte-specific latex labelling procedure. In situ efferocytosis was assessed on mouse aortic root sections. Additionally, metabolic status, macrophage motility, efferocytosis, and involved mechanisms were analysed in peritoneal macrophages. KEY RESULTS: Global heterozygous or myeloid cell-specific Pfkfb3 deficiency reduced atherogenesis in Apoe-/- mice. Mechanistic studies showed that PFKFB3 controlled the proliferation and infiltration of proinflammatory monocytes. Moreover, PFKFB3 expression was associated with inflammatory monocyte expansion in patients with atherosclerotic coronary artery disease. Surprisingly, homozygous loss of Pfkfb3 impaired macrophage efferocytosis and exacerbated atherosclerosis in Apoe-/- mice. Mechanistically, PFKFB3-driven glycolysis was shown to be essential for actin polymerization, thus aiding the efferocytotic function of macrophages. CONCLUSION AND IMPLICATIONS: Collectively, these findings suggest the existence of a double-edged sword effect of myeloid PFKFB3 on the pathogenesis of atherosclerosis and highlight the need for caution in developing anti-atherosclerotic strategies that target PFKFB3.

Reduced expression of netrin-1 is associated with fetal growth restriction.

The objective of this study was to investigate the expression of netrin-1 in placenta from patients with fetal growth restriction (FGR) and its effect on the viability and apoptosis of human placental microvascular endothelial cells. Thirty-three patients with FGR (including eighteen severe cases) and twenty-four normal late pregnant women were investigated. The expression of netrin-1 in placental tissues was detected by employing immunohistochemistry, real-time PCR, and Western blotting. Human placental microvascular endothelial cells were isolated and, after treatment with netrin-1, examined for their viability and apoptosis by using MTT assay and flow cytometry. We demonstrated that the netrin-1 was present in placenta. Netrin-1 was significantly reduced in pregnant women with FGR as compared with the controls. Furthermore, netrin-1 enhanced the viability of human placental microvascular endothelial cells and inhibited their apoptosis. Netrin-1 may regulate the development of placental vessels and plays a key role in the pathogenesis of FGR.

Catalpol stimulates VEGF production via the JAK2/STAT3 pathway to improve angiogenesis in rats' stroke model.

ETHNOBOTANICAL RELEVANCE: Catalpol is the main active component of the radix from Rehmannia glutinosa Libosch, which has pleiotropic protective effects in neurodegenerative diseases, ischemic stroke, metabolic disorders and others AIM: Catalpol has been shown to have neuroprotective, neurorepair, and angiogenesis effects following ischemic brain injury. However, its molecular mechanisms are still poorly understood. In previous studies, the JAK2/STAT3 signaling pathway was found to play a role in neuroprotection and angiogenesis. This study investigated the role of catalpol in stimulating angiogenesis via the JAK2/STAT3 pathway after permanent focal cerebral ischemia (pMCAO). METHODS: Rats were subjected to right middle cerebral artery occlusion through electrocoagulation and were treated with catalpol (5mg/kg), AG490 was also used to inhibit STAT3 phosphorylation (pSTAT3). RESULTS: Following stroke, Catalpol improved the neuroethology deficit, increased the cerebral blood flow (CBF) of infarcted brain and upregulated EPO and EPOR. AG490 suppressed the phosphorylation of signal transducer and activator of transcription 3 (STAT3), ultimately inhibited VEGF mRNA expression, which reduced VEGF protein expression and inhibited stroke-induced angiogenesis. However, Catalpol enhanced stroke-induced STAT3 activation and subsequently restored STAT3 activity through the recovery of STAT3 binding to VEGF. Moreover, Catalpol reversed the effect of AG490 on STAT3 activation and nuclear translocation, restored the transcriptional activity of the VEGF promoter by recruiting STAT3 to the VEGF promoter, improved VEGF mRNA and protein expression, increased angiogenesis, reduced the difference in CBF between the infarcted and intact brain and ameliorated the neuroethology behaviors after stroke. CONCLUSION: Catalpol affects neuroprotection and angiogenesis via the JAK2/STAT3 signaling pathway, which is mediated by STAT3 activation and VEGF expression. Catalpol may be used as a potential therapeutic drug for stroke.

[Regulation of UC-MSC on Immune Inflammatory Thrombophilia in MRL/lpr Mice].

OBJECTIVE: To study the immune repair effect of umbilical cord mesenchymal stem cells (UC-MSC) on inflammatory disorders and thrombophilia state of MRL/lpr mice by detecting the expression change of peripheral blood CD4(+) CD25(+) T cells and the levels of plasma inflammatory cytokines TNF-α, IL-6 and thrombosis indicators TF, FIB. METHODS: Twenty five MRL/lpr mice were divided into control (C) group, UC-MSC one time treatment (UT1) group and UC-MSC three time treatments (UT3) group. UC-MSC cell suspension was injecled via tail vein and these mice were feeded in SPF environment. The blood samples were taken from the mice every 2 weeks after 16(th) week. FCM was used to detect the expression of CD4(+) CD25(+) T cells in peripheral blood, ELISA assay was used to detect the levels of inflammatory cytokines TNF-α, IL-6 and thrombosis indicators TF, FIB. RESULTS: The expression of peripheral blood CD4(+) CD25(+) T cells in treatment groups increased during 16(th) to 18(th) week, dropped and tended to be stable since 20(th) week, and lower than those in control group. The levels of plasma TNF-α and IL-6 in treatment group decreased since 16(th) week and significantly lower than those in control group (P < 0.05). The levels of plasma TF and FIB in treatment group decreased since 16(th) week. The level of plasma TF in treatment group was significantly lower than those in control group (P < 0.05) since 18(th) week. CONCLUSION: UC-MSC can repair the immune inflammatory microenvironment disorders of MRL/lpr mice through its immunomodulatory effect. UC-MSC contribute to repair of immune inflammatory thrombophilia of MRL/lpr mice.

Human umbilical cord mesenchymal stem cells improve the immune-associated inflammatory and prothrombotic state in collagen type-â ¡-induced arthritic rats.

Human umbilical cord mesenchymal stem cells (hUC­MSCs) hold great potential in the search for therapies to treat refractory diseases, including rheumatoid arthritis (RA), due to their potential regenerative ability and extensive source. However, the role of hUC­MSCs in vivo and the repair mechanisms for RA remain to be fully elucidated. The present study aimed to determine whether hUC­MSCs exert immunomodulatory effects and have anti­inflammatory capabilities in the treatment of embolisms. Following the transplantation of hUC­MSCs into collagen type Ⅱ­induced arthritic (CIA) model rats, magnetic resonance imaging (MRI) in vivo was performed, and the levels of interleukin (IL)­1, IL­17, tumor necrosis factor (TNF)­α, vascular endothelial growth factor (VEGF), tissue factor (TF), CD4+CD25+ T cells (Treg) and antithrombin (AT) were measured. Bromodeoxyuridine staining was performed for histopathological examinations. As revealed by immunofluorescence and MRI experiments, the injected hUC­MSCs preferentially migrated to the inflammatory joint sites of the rats. The Treg cell percentage and AT levels in the hUC­MSC­treated group were markedly increased, whereas the levels of IL­1, IL­17, TNF­α, VEGF and TF were decreased compared with those in the CIA model group. The values determined for these parameters in the hUC­MSC­treated group returned to approximately the identical values as those of the control group on day 35 post­therapy. Superparamagnetic iron oxide nanoparticles (SPIONs) may serve as an effective, non­invasive method for tracking transplanted cells in vivo. The present study provided direct evidence that hUC­MSCs in the CIA rat model migrated to the inflammatory joint sites, effectively promoting recovery from collagen type II damage and thereby improving the immune­associated prothrombotic state.

[Systemic lupus erythematosus and immune-inflammatory thrombophilia].

Systemic lupus erythematosus (SLE) is an autoimmune disease with causes including activation of innate and adaptive immune systems. SLE patients are with a high risk of thrombosis, which may be due to disease activation, immune complexes, toxic antibodies and high level of inflammation. This article discusses neutrophil/NET factor, antibody factor, platelet factor and particle factor which is involved in coagulation pathways and thrombus formation mechanism under the state of immune disorders in SLE.

Square wave anodic stripping voltammetric determination of Cd²âº and Pb²âº at bismuth-film electrode modified with electroreduced graphene oxide-supported thiolated thionine.

Graphene oxide (GO)-thionine (TH) nanocomposite was prepared by π-π stacking. The nanocomposite was cast-coated on a glassy carbon electrode (GCE) to prepare an electroreduced GO (ERGO)-TH/GCE, then 2-mercaptoethanesulfonate (MES) was covalently tethered to ERGO-TH by potentiostatic anodization to form an ERGO-TH-MES/GCE. The thiolation reaction was monitored by electrochemical quartz crystal microbalance (EQCM). Square wave anodic stripping voltammetry (SWASV) was used to determine Cd(2+) and Pb(2+) at the ERGO-TH-MES/GCE further modified with Nafion and Bi. Under the optimal conditions, the linear calibration curves for Cd(2+) and Pb(2+) are from 1 to 40 µg L(-1), with limits of detection (S/N=3) of 0.1 µg L(-1) for Cd(2+) and 0.05 µg L(-1) for Pb(2+), respectively. The electrode was used for the simultaneous analysis of Cd(2+) and Pb(2+) in water samples with satisfactory recovery.

[Immunomodulatory effect of UC-MSC on function of immunocytes of rats with collagen type II induced arthritis].

This study was purposed to observe the influence of umbilical cord mesenchymal stem cells (UC-MSC) on the peripheral blood CD4(+)CD25(+)regulatory T cells (Treg), Th17 cells and neutrophils in rats with collagen type II-induced arthritis(CIA), and to explore the regulating effect of UC-MSC transplantation on immunocyte subgroup. The rats wee divided into 3 groups: CIA group (model group), UC-MSC treated group and blank control group. The CIA rats were injected with UC-MSC via tail vein. The percentage of CD4(+)CD25(+) cells in peripheral blood and the expression of NCD11b on neutrophil surface in CIA rates was detected by flow cytometry (FCM), and the serum interleukin-17 (IL-17) was observed by enzyme-linked immunosorbent assay (ELISA). The results showed that the mean fluorescence intensity(MFI) of NCD11b and the level of IL-17 in the model group were significantly higher than those in the blank control group, and the ratio of CD4(+)CD25(+) cells were significantly lower (P < 0.05). The MIF of NCD11b and the level of IL-17 in the UC-MSC treated group were significantly lower than that in the model group (P < 0.05), while the proportion of CD4(+)CD25(+) Treg increased (P < 0.05). Since the fifth week, the above indicators in the UC-MSC group have almostly approached the control group. It is concluded that the UC-MSC can increase peripheral blood Treg proportion in CIA rat, inhibit the secretion of Th17 and the activity of neutrophils, reduce the immune inflammation reaction, decrease the release of proinflammatory factor, and induce immune reconstruction.

Horseradish peroxidase-catalyzed synthesis of poly(thiophene-3-boronic acid) biocomposites for mono-/bi-enzyme immobilization and amperometric biosensing.

We report here on a facile enzymatic polymerization protocol to prepare enzyme-poly(thiophene-3-boronic acid) (PTBA) polymeric biocomposites (PBCs) for high-performance mono-/bi-enzyme amperometric biosensing. Horseradish peroxidase (HRP)-catalyzed polymerization of thiophene-3-boronic acid (TBA) monomer was conducted in aqueous solution containing HRP (or plus glucose oxidase (GOx)) by either directly added or GOx-glucose generated oxidant H2O2. The mono-/bi-enzyme amperometric biosensors were prepared simply by casting the dialysis-isolated PBCs on Au-plated Au electrode (Auplate/Au), followed by coating with an outer-layer chitosan (CS) film. The boronic acid residues are capable of covalent bonding with enzyme at the glycosyl sites (boronic acid-diols interaction), which should less affect the enzymatic activity as compared with the common cases of covalent bonding at the peptide chains, and UV-vis spectrophotometric tests confirmed that the encapsulated HRP almost possesses its pristine enzymatic specific activity. The enzyme electrodes were studied by cyclic voltammetry, electrochemical impedance spectroscopy and chronoamperometry in the presence of Fe(CN)6(4-) mediator. The CS/HRP-PTBA/Auplate/Au electrode responded linearly to H2O2 concentration from 1 to 300 µM with a sensitivity of 390 µA mM(-1)cm(-2) and a limit of detection (LOD) of 0.1 µM. The bienzyme CS/GOx-HRP-PTBA(H2O2)/Auplate/Au electrode responded linearly to glucose concentration from 5 µM to 0.83 mM with a sensitivity of 75.1 µA mM(-1)cm(-2) and a LOD of 1 µM, and it is found here that the use of Fe(CN)6(4-) that can only efficiently mediate HRP favorably avoids the "unusual amperometric responses" observed when other mediators that can efficiently turn over both HRP and GOx are used.

Fabrication of a chitosan/glucose oxidase-poly(anilineboronic acid)-Au(nano)/Au-plated Au electrode for biosensor and biofuel cell.

Enzyme immobilization is one of the key factors in constructing high-performance enzyme biosensors and biofuel cells (BFCs). Herein, we propose a new protocol for efficient immobilization of a glycoprotein enzyme based on the interaction of the 1, 2- or 1, 3-diols in the glycoprotein with a boronic acid functionalized monomer. Briefly, casting a mixture of glucose oxidase (GOx) and anilineboronic acid (ABA) followed by a NaAuCl(4) solution to an Au-plated Au electrode surface yielded a GOx-poly(ABA) (PABA)-gold nanoparticle (Au(nano)) bionanocomposite, and chitosan (CS) was then cast and air-dried. In the present protocol, the small-sized Au(nano) or Au subnanostructures can form near/on the enzyme molecule, which greatly promotes the electron transfer of enzymatic reaction and enhances the amperometric responses. The thus-prepared CS/GOx-PABA-Au(nano)/Au-plated Au electrode worked well in the first-/second generation biosensing modes and as a bioanode in a monopolar biofuel cell, with analytical or cell-power performance superior to those of most analogues hitherto reported.