Maximum Reaction Velocity Research Articles

Axial Chlorination Engineering of Single-Atom Nanozyme: Fe-N4Cl Catalytic Sites for Efficient Peroxidase-Mimicking.

Developing axial coordination engineering of single-atom nanozymes (SAzymes), directly regulating the axial coordination environment of the catalytic site, and optimizing the axial adsorption are meaningful and challenging for boosting the enzyme-like activities. Herein, the axial chlorination engineering of SAzyme with the Fe-N4Cl catalytic site (Fe-N4Cl/CNCl) was first proposed, exhibiting superior peroxidase-like activity compared to the traditional Fe-N4/CN SAzyme with Fe-N4 site. The maximal reaction velocity (4.73 × 10-5 M min-1), the catalytic constant (246.4 min-1), and the specific activity (81 U/mg) catalyzed by the Fe-N4Cl/CNCl SAzyme were 4.9 times, 3.9 times, and 2.7 times those of the Fe-N4/CN SAzyme, revealing the enormous advantages of axial chlorination engineering of SAzymes for remarkably improving enzyme-like activities. Moreover, the Fe-N4Cl/CNCl SAzyme also exhibited an enhanced inhibition effect of tumor cell growth in vitro and in vivo. The density functional theory calculation revealed that the Fe-N4Cl site was more favorable for releasing •OH radical, lowering the energy barrier of rate-determining step, and accelerating the reaction rate compared to the Fe-N4 site. This work demonstrated the outstanding potential of axial chlorination engineering of SAzymes for improving enzyme-like activities and practical application in tumor therapy.

Biochemical characterization of HcrF from Limosilactobacillus fermentum, a NADH-dependent 2-ene reductase with activity on hydroxycinnamic acids.

In fermented plant foods, phenolic compounds are metabolized by 2-ene reductases, which reduce double bonds adjacent to an aromatic rings in phytochemicals including hydroxycinnamic acids, isoflavones, and flavones. Only few 2-ene reductases of lactic acid bacteria were characterized, including the hydrocinnamic reductases HcrB and Par1, and the daidzein reductase of Lactococcus lactis. This study aimed to characterize HcrF, a homologue of HcrB, in Limosilactobacillus fermentum. HcrF was purified after cloning in Escherichia coli and purification by affinity chromatography. HcrF was optimally active at 30 - 40°C and pH 7.0 and required both FMN and NADH as co-factors. Ferulic, caffeic, p-coumaric and sinapic acids but not trans-cinnamic acids were reduced to dihydro derivatives. The maximum reaction velocity Vmax of HcrF was highest for ferulic acid. On a phylogenetic tree of 2-ene reductases, HcrF clustered most closely with the hydroxycinnamic acid reductase HcrB of Lactiplantibacillus plantarum. The hydroxycinnamic acid reductase Par1 of Furfurilactobacillus milii and flavone or isoflavone reductases were only distantly related to HcrF. In summary, current knowledge does not allow to predict the substrate specificity of 2-ene reductases on the basis of the protein sequence; this study adds HcrF to the short list of enzymes with known substrate specificity.

Gas Chromatography-Mass Spectrometry and Fourier Transform Infrared Spectroscopy Analysis of Potential α-Glucosidase Inhibitors from Terminalia catappa Leaf Extracts

Aims: Postprandial hyperglyceamia is often caused by insulin insufficiency or cellular glucose uptake complications, and conventional treatments often yield undesirable side effects. The aim of the current work was to assess the α-glucosidase inhibitory activities of T. catappa leaf extracts and use spectroscopic techniques to partially characterize possible inhibitors. Study Design: Phytochemical screening, enzyme inhibition assay and spectrometric and spectroscopic characterization of bioactive compounds from leaf extract and fractions of T. catappa. Place and Duration of Study: The entire work was carried out at the Department of Applied Chemistry and Biochemistry, University for Development Studies, Ghana within nine months. Methodology: T. catappa leaf extract and fractions were qualitatively screened for phytochemicals and their biological activity assessed in α-glucosidase inhibition assay. The potential enzyme inhibitors were characterized by Gas Chromatography-Mass Spectrometry (GC-MS) and Fourier Transform Infrared (FT-IR) Spectroscopy. Results: Phytochemical screening of the extract and the various solvent fractions revealed the presence of alkaloids, flavonoids, saponins, flavanol glycosides, phenolics, and terpenoids. The α-glucosidase inhibition potential of the crude leaf extract was concentration-dependent with a half-maximal inhibitory concentration (IC50) of 337.5 µg/ml. Solvent fractions from the crude extract demonstrated α-glucosidase inhibitory activity with IC50 values ranging from 170.40 µg/ml for ethyl acetate (EtOAc) fraction to 71.90 µg/ml for n-hexane (n-Hex) fraction. Acarbose, the control drug, demonstrated significant α-glucosidase inhibition activity in a similar pattern with IC50 of 6.16 µg/ml. The enzyme inhibition kinetics parameters, specifically the Michaelis constant (KM) and Maximum reaction velocity (Vmax) values, suggested that the inhibition of α-glucosidase by T. catappa extract followed a competitive mode. GC-MS and FT-IR analysis of the n-Hexane fraction identified the compounds Eugenol, Urs-12-en-24-oic acid, 3-oxo-, methyl ester (+)-, phytol, trans-isoeugenol, α-amyrin, and squalene as the potential antidiabetic agents that might be responsible for reducing postprandial blood glucose levels. Conclusion: These findings show that T. catappa leaf extract contains α-glucosidase inhibitors and therefore serves as a valuable resource in the discovery of natural anti-diabetic agents.

Identification and characterization of Lacticaseibacillus rhamnosus HP-B1083-derived β-glucuronidase and its application for baicalin biotransformation

Baicalein, showing higher bioavailability and stronger pharmacological activity, can be obtained via a β-glucuronidase (GUS)-catalyzed transformation of baicalein 7-O-β-D-glucuronide (baicalin). Recently, we have found that the fermentation broth of Lacticaseibacillus rhamnosus HP-B1083 can efficiently convert baicalin to baicalein. In this study, the L. rhamnosus HP-B1083-derived enzyme involved in baicalin biotransformation was identified and characterized. First, the LruidA gene, encoding the responsible enzyme, was cloned and sequenced. Sequence analysis revealed that the deduced enzyme (designated as LrUidA) belonged to the glycosyl hydrolase family 2. The recombinant LrUidA was expressed and purified for characterization. LrUidA had a molecular weight of 70 kDa, with an optimal temperature of 50 °C and pH 4.5. Although LrUidA was susceptible to temperature, it possessed a relative pH stability. Its Michaelis–Menten constant, maximum reaction velocity and catalytic constant values were 9.710 mM, 13.08 mM/min/mg, and 14.95 s−1, respectively. Site-directed mutagenesis experiment results demonstrated that the enzyme reaction uses side chains of E509 and E415 to hydrolyze the glycosidic bond of baicalin and involves three negatively charged residues, E450, D451, and D452, respectively. Surprisingly, biotransformation was performed under optimized reaction conditions by incubating the purified enzyme with 0.1 % baicalin for 4 h, resulting in a considerable conversion ratio of 99 %. Altogether, our findings provide insights into the properties of L. rhamnosus HP-B1083-derived enzyme and expand our understanding regarding using GUS for the industrial production of baicalein.

Fluorescent Mn3O4 quantum dot as catechol oxidase nanozyme: A robust nano-platform for sensitive dopamine detection

The development of multifunctional semi-conductor nano-enzymes has attained significant interest recently, due to their ease in fabrication, cost-effectiveness, and wide range of applications. In this work, we report the synthesis of ammine-functionalized Mn3O4 quantum dots (Mn3O4 QDs) with excellent luminescence properties. These QDs have been developed via the reduction of permanganate using L-cysteine through a simple, facile one-step bottom-up approach. The steady-state enzyme kinetic studies revealed the superior Catechol-Oxidase mimic activity of the Mn3O4 QDs with a Michaelis-Menten constant (Km) of 133.19 µM, and maximum reaction velocity (Vmax) of 69.40 µMs−1. Due to this superior enzymatic activity, dopamine (DA) gets easily oxidized into DA-quinone and eventually polymerized to form polydopamine. Schiff base/Michael addition reaction between nucleophilic ammine on the surface of QDs and the aromatic chain makes the probe and analyte come closer and leads to the formation of a charge transfer complex, resulting in fluorescence quenching of QDs. This fluorescence quenching has been quantified to develop a turn-off sensor for DA. The sensing characteristics are highly selective and sensitive towards DA with a detection limit of 18 nM. The synthesised Mn3O4 QDs with superior enzyme-mimicking properties have tremendous potential as fluorescent probes in imaging and health care applications.

Atom-pair engineering of single-atom nanozyme for boosting peroxidase-like activity

Constructing atom-pair engineering and improving the activity of metal single-atom nanozyme (SAzyme) is significant but challenging. Herein, we design the atom-pair engineering of Zn-SA/CNCl SAzyme by simultaneously constructing Zn-N4 sites as catalytic sites and Zn-N4Cl1 sites as catalytic regulator. The Zn-N4Cl1 catalytic regulators effectively boost the peroxidase-like activities of Zn-N4 catalytic sites, resulting in a 346-fold, 1496-fold, and 133-fold increase in the maximal reaction velocity, the catalytic constant and the catalytic efficiency, compared to Zn-SA/CN SAzyme without the Zn-N4Cl1 catalytic regulator. The Zn-SA/CNCl SAzyme with excellent peroxidase-like activity effectively inhibits tumor cell growth in vitro and in vivo. The density functional theory (DFT) calculations reveal that the Zn-N4Cl1 catalytic regulators facilitate the adsorption of *H2O2 and re-exposure of Zn-N4 catalytic sites, and thus improve the reaction rate. This work provides a rational and effective strategy for improving the peroxidase-like activity of metal SAzyme by atom-pair engineering.

High catalytic nickel−platinum nanozyme enhancing colorimetric detection of Salmonella Typhimurium in milk

Colorimetric qualitative and sensitive quantitative detection of Salmonella Typhimurium (S. Typhimurium) holds significant importance for ensuring food safety and preventing foodborne illnesses. In the study, an ultra-high catalytic activity and biocompatible nickel-platinum nanoparticle (NiPt NP) nanozyme is successful synthesized to prepare a NLISA strategy for the detection of S. Typhimurium. The synthesized NiPt NPs exhibit high oxidase-like catalytic efficiency, with a Michaelis constant (Km) of 0.493 mM, similar to that of natural horseradish peroxidase (HRP). The maximal reaction velocity (Vmax) was determined to be 1.97 × 10-7 M·s-1 exhibiting a 1.97-fold higher than that of the HRP (1.0 × 10-7 M·s-1). Meanwhile, the antibody employed in this NiPt NPs-based NLISA exhibits exceptional capture efficacy, generating a stable immune complex with S. Typhimurium. The NiPt NPs-based NLISA demonstrates sensitivity, specificity, convenience, and cost-efficiency for the detection of S. Typhimurium. Under optimal conditions, this NiPt NPs-based NLISA demonstrates a quantitative range of 103∼106 cfu/mL with a detection limit as low as 103 cfu/mL. A single-blind experimental testing detects different concentrations of S. Typhimurium spiked skim milk, indicating the application potential of the proposed NLISA in real samples. In all, this research provides novel insights into the synthesis of nanozymes with excellent catalytic activity and their applications in S. Typhimurium biosensing.

Copper-based metal-organic frameworks (BDC-Cu MOFs) as supporters for α-amylase: Stability, reusability, and antioxidant potential

Copper-based metal-organic frameworks (BDC-Cu MOFs) were synthesized via a casting approach using 1,4-benzene dicarboxylic (BDC) as organic ligand and their properties characterized. The obtained materials were then utilized to immobilize the α-amylase enzyme. The chemical composition and functional components of the synthesized support (BDC-Cu MOFs) were investigated with Fourier transform infrared spectroscopy (FTIR), the surface morphology was determined with scanning electron microscopy (SEM), and the elemental composition was established with energy dispersive X-ray (EDX) analyses. X-ray diffraction (XRD) was employed to analyze the crystallinity of the synthesized DBC-Cu MOFs. The zeta potentials of DBC-Cu MOFs and DBC-Cu MOFs@α-amylase were determined. The immobilized α-amylase demonstrated improved catalytic activity and reusability compared to the free form. Covalent attachment of the α-amylase to BDC-Cu provided an immobilization yield (IY%) of 81% and an activity yield (AY%) of 89%. The immobilized α-amylase showed high catalytic activity and 81% retention even after ten cycles. Storage at 4 °C for eight weeks resulted in a 78% activity retention rate for DBC-Cu MOFs@α-amylase and 49% retention for the free α-amylase. The optimum activity occurred at 60 °C for the immobilized form, whereas the free form showed optimal activity at 50 °C. The free and immobilized α-amylase demonstrated peak catalytic activities at pH 6.0. The maximum reaction velocities (Vmax) values were 0.61 U/mg of protein for free α-amylase and 0.37 U/mg of protein for BDC-Cu MOFs@α-amylase, while the Michaelis‒Menten affinity constants (Km) value was lower for the immobilized form (5.46 mM) than for the free form (11.67 mM). Treatments of maize flour and finger millet samples with free and immobilized α-amylase resulted in increased total phenolic contents. The enhanced antioxidant activities of the treated samples were demonstrated with decreased IC50 values in ABTS and DPPH assays. Overall, immobilization of α-amylase on BDC-Cu MOFs provided improved stability and catalytic activity and enhanced the antioxidant potentials of maize flour and finger millet.

Nanozyme-based inulin@nanogold for adhesive and antibacterial agent with enhanced biosafety

Nanozymes with oxidase or peroxidase-mimicking activity have emerged as a promising alternative for disinfecting resistant pathogens. However, further research and clinical applications of nanozymes are hampered by their low in vivo biosafety and biocompatibility. In this study, inulin-confined gold nanoparticles (IN@AuNP) are synthesized as an antibacterial agent via a straightforward in situ reduction of Au3+ ions by the hydroxyl groups in inulin. The IN@AuNP exhibits both peroxidase-mimicking and oxidase-mimicking catalytic activities, of which the maximum reaction velocity (Vmax) for H2O2 is 2.66 times higher than that of horseradish peroxidase. IN@AuNP can catalyze the production of reactive oxygen species (ROS), resulting in effective antibacterial behavior against both Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria. Abundant hydroxyl groups retained in inulin endow the nanozyme with high adhesion to bacteria, reducing the distance between the captured bacteria and ROS, achieving an antibacterial ratio of 100 % within 1 h. Importantly, due to the natural biosafety and non-absorption of the dietary fiber inulin, as well as the inability of inulin-trapped AuNP to diffuse, the IN@AuNP exhibits high biosafety and biocompatibility under physiological conditions. This work is expected to open a new avenue for nanozymes with great clinical application value.

Theory on the rate equations of Michaelis-Menten type enzyme kinetics with competitive inhibition.

We derive approximate expressions for pre- and post-steady state regimes of the velocity-substrate-inhibitor spaces of the Michaelis-Menten enzyme kinetic scheme with fully and partial competitive inhibition. Our refinement over the currently available standard quasi steady state approximation (sQSSA) seems to be valid over wide range of enzyme to substrate and enzyme to inhibitor concentration ratios. Further, we show that the enzyme-inhibitor-substrate system can exhibit temporally well-separated two different steady states with respect to both enzyme-substrate and enzyme-inhibitor complexes under certain conditions. We define the ratios fS = vmax/(KMS + e0) and fI = umax/(KMI + e0) as the acceleration factors with respect to the catalytic conversion of substrate and inhibitor into their respective products. Here KMS and KMI are the Michaelis-Menten parameters associated respectively with the binding of substrate and inhibitor with the enzyme, vmax and umax are the respective maximum reaction velocities and e0, s0, and i0 are total enzyme, substrate and inhibitor levels. When (fS/fI) < 1, then enzyme-substrate complex will show multiple steady states and it reaches the full-fledged steady state only after the depletion of enzyme-inhibitor complex. When (fS/fI) > 1, then the enzyme-inhibitor complex will show multiple steady states and it reaches the full-fledged steady state only after the depletion of enzyme-substrate complex. This multi steady-state behavior especially when (fS/fI) ≠ 1 is the root cause of large amount of error in the estimation of various kinetic parameters of fully and partial competitive inhibition schemes using sQSSA. Remarkably, we show that our refined expressions for the reaction velocities over enzyme-substrate-inhibitor space can control this error more significantly than the currently available sQSSA expressions.

Evaluation of arsenic pollution in field-contaminated soil at the soil's actual pH

The pollution and ecological risks posed by arsenic (As) entering the soil are the major environmental challenges faced by human beings. Soil phosphatase can serve as a useful indicator for assessing As contamination under specific soil pH conditions. However, the study of phosphatase kinetics in long-term field As-contaminated soil remains unclear, presenting a significant obstacle to the monitoring and evaluation of As pollution and toxicity. The purpose of this study was to determine phosphatase activity and explore enzyme kinetics in soils subjected to long-term field As contamination. Results revealed that the soil phosphatase activity varied among the tested soil samples, depending on the concentrations of As. The relationship between total As, As fractions and phosphatase activity was found to be significant through negative exponential function fitting. Kinetic parameters, including maximum reaction velocity (Vmax), Michaelis constant (Km) and catalytic efficiency (Vmax/Km), ranged from 3.14 × 10−2–53.88 × 10−2 mmol/(L·hr), 0.61–7.92 mmol/L, and 0.46 × 10−2–11.20 × 10−2 hr−1, respectively. Vmax and Vmax/Km of phosphatase decreased with increasing As pollution, while Km was less affected. Interestingly, Vmax/Km showed a significant negative correlation with all As fractions and total As. The ecological doses (ED10) for the complete inhibition and partial inhibition models ranged from 0.22–70.33 mg/kg and 0.001–55.27 mg/kg, respectively, indicating that Vmax/Km can be used as an index for assessing As pollution in field-contaminated soil. This study demonstrated that the phosphatase kinetics parameters in the soil's pH system were better indicators than the optimal pH for evaluating the field ecotoxicity of As.

Computational Modeling of Substrate-Dependent Mitochondrial Respiration and Bioenergetics in the Heart and Kidney Cortex and Outer Medulla.

Integrated computational modeling provides a mechanistic and quantitative framework to characterize alterations in mitochondrial respiration and bioenergetics in response to different metabolic substrates in-silico. These alterations play critical roles in the pathogenesis of diseases affecting metabolically active organs such as heart and kidney. Therefore, the present study aimed to develop and validate thermodynamically constrained integrated computational models of mitochondrial respiration and bioenergetics in the heart and kidney cortex and outer medulla (OM). The models incorporated the kinetics of major biochemical reactions and transport processes as well as regulatory mechanisms in the mitochondria of these tissues. Intrinsic model parameters such as Michaelis-Menten constants were fixed at previously estimated values, while extrinsic model parameters such as maximal reaction and transport velocities were estimated separately for each tissue. This was achieved by fitting the model solutions to our recently published respirometry data measured in isolated rat heart and kidney cortex and OM mitochondria utilizing various NADH- and FADH2-linked metabolic substrates. The models were validated by predicting additional respirometry and bioenergetics data, which were not used for estimating the extrinsic model parameters. The models were able to predict tissue-specific and substrate-dependent mitochondrial emergent metabolic system properties such as redox states, enzyme and transporter fluxes, metabolite concentrations, membrane potential, and respiratory control index under diverse physiological and pathological conditions. The models were also able to quantitatively characterize differential regulations of NADH- and FADH2-linked metabolic pathways, which contribute differently toward regulations of oxidative phosphorylation and ATP synthesis in the heart and kidney cortex and OM mitochondria.

CuO@AgNPs nanozyme cavity arrays on screen-printed electrodes for ultrasensitive and on-site SERS detection

The research on the use of metal–semiconductor hybrid systems for constructing chemical and biological sensors has received increasing attention. In this work, a screen-printed electrode (SPE) with peroxidase activity was developed using AgNPs decorated CuO cavity array (CuO@Ag SPE) for on-site SERS detection of Leuco crystal violet (LCV) in aquatic products. The low Michaelis-Menten constant (Km = 42.8 μM) and the high maximum reaction velocity (Vm = 5.1 × 10−8 mol·L−1s−1) indicate that the CuO@Ag SPE has a strong affinity and excellent catalytic efficiency. Moreover, this substrate has a considerable SERS enhancement with EF of 6.1 × 106, resulting from the synergetic effect of the electromagnetic enhancement (EM) and chemical enhancement (CM) after modifying noble metal onto semiconductor materials. The CuO@Ag SPE can efficiently catalyze the oxidation of the Raman-inactive reporter (LCV) and simultaneously act as SERS-active substrates for detecting the generated Raman-active crystal violet (CV), providing a relatively wide linear range (2.0 nM-2.0 μM) and a lower detection limit (0.3 nM). The recoveries obtained from the proposed SPE range from 100.1 to 109.2% and RSD from 4.9 to 10.4%, demonstrating that the SPE has high accuracy and reliability in real sample detection.

A NIR-II Photoactivatable "ROS Bomb" with High-Density Cu2 O-Supported MoS2 Nanoflowers for Anticancer Therapy.

The fast conversion of hydrogen peroxide (H2 O2 )into reactive oxygen species (ROS) at tumor sites is a promising anticancer strategy by manipulating nanomedicines with near-infrared light in the second region (NIR-II). However, this strategy is greatly compromised by the powerful antioxidant capacity of tumors and the limited ROS generation rate of nanomedicines. This dilemma mainly stems from the lack of an effective synthesis method to support high-density copper-based nanocatalysts on the surface of photothermal nanomaterials. Herein, a multifunctional nanoplatform (MCPQZ) with high-density cuprous (Cu2 O) supported molybdenum disulfide (MoS2 ) nanoflowers (MC NFs) is developed for the efficient killing of tumors via a potent ROS storm by an innovative method. Under NIR-II light irradiation, the ROS intensity and maximum reaction velocity (Vmax ) produced by MC NFs are21.6and 33.8times that of the non-irradiation groupin vitro, which is much higher than most current nanomedicines. Moreover, the strong ROS storm in cancer cells isefficiently formed by MCPQZ (increased by 27.8times compared to the control), thanks to the fact that MCPQZ effectively pre-weakens the multiple antioxidant systems of cancer cells. This work provides a novel insight to solve the bottleneck of ROS-based cancer therapy.

Cubic Ag2O nanoparticles as robust laccase mimetics in a smartphone-assisted colorimetric sensor for rapid and ultrasensitive detection of kanamycin in environment

Due to the excellent oxidation effect on phenolic compounds, the present copper-based laccase-mimics are mostly used for the degradation and detection of phenolic pollutants in environment. As the oxidation substrates of laccase are only limited to compounds containing specific phenolic hydroxyl groups, which makes the application of laccase mimetics to non-phenolic pollutants faces great challenges and bottlenecks. Herein, a non-copper-based laccase mimics like cubic Ag2O nanoparticles is prepared. The maximum reaction velocity and affinity of cubic Ag2O nanoparticles to substrates are significantly better than that of natural laccase and most artificial laccase mimetics. Cubic Ag2O nanoparticles also exhibits high stability under various conditions and has good tolerance to organic solvents. Moreover, it is found that the laccase-mimic activity of cubic Ag2O nanoparticles can be effectively inhibited by kanamycin, which enable us to construct a colorimetric sensor for rapid detection of kanamycin in environment. The sensor has excellent sensitivity with the limit of detection (LOD) of 1.3 nM for kanamycin, and has high selectivity for other competitive substances. Besides, a standard curve for rapidly detecting kanamycin by smartphones is established, which expect to provide a novel idea for on-site monitoring of other hazards in environment.

Synergistic inhibitory effects of clopidogrel and rivaroxaban on platelet function and platelet-dependent thrombin generation in cats.

Dual antithrombotic treatment (DAT) with clopidogrel and rivaroxaban sometimes is prescribed to cats with hypertrophic cardiomyopathy at risk of thromboembolism. To date, no studies have evaluated their combined effects on platelet function. Evaluate the safety of DAT in healthy cats and compare, ex vivo, platelet-dependent thrombin generation and agonist-induced platelet activation and aggregation in cats treated with clopidogrel, rivaroxaban, or DAT. We hypothesized that DAT would safely modulate agonist-induced platelet activation and aggregation more effectively than single agent treatment. Nine apparently healthy 1-year-old cats selected from a research colony. Unblinded, nonrandomized ex vivo cross-over study. All cats received 7 days of rivaroxaban (0.6 ± 0.1 mg/kg PO), clopidogrel (4.7 ± 0.8 mg/kg PO), or DAT with defined washout periods between treatments. Before and after each treatment, adenosine diphosphate (ADP)- and thrombin-induced platelet P-selectin expression was evaluated using flow cytometry to assess platelet activation. Platelet-dependent thrombin generation was measured by fluorescence assay. Platelet aggregation was assessed using whole blood impedance platelet aggregometry. No cats exhibited adverse effects. Of the 3 treatments, only DAT significantly decreased the number of activated platelets (P = .002), modulated platelet activation in response to thrombin (P = .01), dampened thrombin generation potential (P = .01), and delayed maximum reaction velocity (P = .004) in thrombin generation. Like clopidogrel, DAT inhibited ADP-mediated platelet aggregation. However, rivaroxaban alone resulted in increased aggregation and activation in response to ADP. Treatment combining clopidogrel and rivaroxaban (DAT) safely decreases platelet activation, platelet response to agonists, and thrombin generation in feline platelets more effectively than monotherapy with either clopidogrel or rivaroxaban.

Kinetic Hydrolysis of Cellulose Biopolymer by Carbon Nanotubes Immobilized Cellulase

Immobilized enzymes are widely used in various biochemical reactions due to higher stability and reusability. Immobilization enzyme is a process of confinement enzyme molecules onto/within a support or matrix material via the chemical or physical process. In this study, the effect of free and CNT-Immobilized cellulase was investigated for hydrolysis of different CMC concentrations. The effect of free and CNT-Immobilized cellulase on the kinetic parameters Vmax and Km in the CMC hydrolysis was compared. The maximum reaction velocity Vmax of CNT-Immobilized cellulase is similar to free cellulase which indicates cellulase adsorption in CNT is effective in hydrolyzing CMC. However, the observed Km values for CNT-Immobilized cellulase is higher than free cellulase for different CMC concentrations. The Km value for CNT-Immobilized shows a sharp decrease in substrate affinity as compared to free cellulase due to diffusional restrictions by the CNT matrix. In addition, the initial velocity (V0) of CMC hydrolysis by CNT-Immobilized cellulase shows similar increasing pattern with the increase of reaction mixture viscosity. The current preparation of CNT-Immobilized cellulase have almost the same catalytic reaction in CMC hydrolysis as free cellulase. Thus, the current preparation of CNT-Immobilized has significant potential for green and sustainable biocatalyst in cellulose-based biopolymer hydrolysis reaction.

Effect of mutations on acetohydroxyacid synthase (AHAS) function in Cyperus difformis L.

Cyperus difformis L. is a troublesome weed in paddy fields and has attracted attention due to its resistance to acetohydroxyacid synthase (AHAS) inhibitors. It was found that the amino acid mutation in AHAS was the primary cause for the resistance of Cyperus difformis. However, the effect of different mutations on AHAS function is not clear in Cyperus difformis. To confirm the effect of mutations on AHAS function, six biotypes were collected, including Pro197Arg, Pro197Ser, Pro197Leu, Asp376Glu, Trp574Leu and wild type, from Hunan, Anhui, Jiangxi and Jiangsu provinces, China and the function of AHAS was characterized. The AHAS in vitro inhibition assay results indicated that the mutations decreased the sensitivity of AHAS to pyrazosulfuron-ethyl, in which the I50 (the half maximal inhibitory concentration) of wild type AHAS was 0.04 μmol L–1 and Asp376Glu, Pro197Leu, Pro197Arg, Pro197Ser and Trp574Leu mutations were 3.98, 11.50, 40.38, 38.19 and 311.43 μmol L–1, respectively. In the determination of enzyme kinetics parameters, the Km and the maximum reaction velocity (Vmax) of the wild type were 5.18 mmol L–1 and 0.12 nmol mg–1 min–1, respectively, and the Km values of AHAS with Asp376Glu, Trp574Leu, Pro197Leu and Pro197Ser mutations were 0.38–0.93 times of the wild type. The Km value of the Pro197Arg mutation was 1.14 times of the wild type, and the Vmax values of the five mutations were 1.17–3.33-fold compared to the wild type. It was found that the mutations increased the affinity of AHAS to the substrate, except for the Pro197Arg mutation. At a concentration of 0.0032–100 mmol L–1 branched-chain amino acids (BCAAs), the sensitivity of the other four mutant AHAS biotypes to feedback inhibition decreased, except for the Pro197Arg mutation. This study elucidated the effect of different mutations on AHAS function in Cyperus difformis and provided ideas for further study of resistance development.

Platinum-Nickel Nanoparticles with Enhanced Oxidase-like Activity for Total Antioxidant Capacity Bioassay.

While great progress in nanozyme-enabled analytical chemistry has been made, most current nanozyme-based biosensing platforms are based on peroxidase-like nanozymes. However, peroxidase-like nanozymes with multienzymatic activities can influence the detection sensitivity and accuracy, while the use of unstable hydrogen peroxide (H2O2) in a peroxidase-like catalytic reaction may result in the reproducibility challenge of sensing signals. We envision that constructing biosensing systems by using oxidase-like nanozymes can address these limitations. Herein, we reported that platinum-nickel nanoparticles (Pt-Ni NPs) with Pt-rich shells and Ni-rich cores possessed high oxidase-like catalytic efficiency, exhibiting a 2.18-fold higher maximal reaction velocity (vmax) than initial pure Pt NPs. The oxidase-like Pt-Ni NPs were applied to develop a colorimetric assay for the determination of total antioxidant capacity (TAC). The antioxidant levels of four bioactive small molecules, two antioxidant nanomaterials, and three cells were successfully measured. Our work not only provides new insights for preparing highly active oxidase-like nanozymes but also manifests their applications for TAC analysis.

Triple-enzyme-mimicking AuPt3Cu hetero-structural alloy nanozymes towards cascade reactions in chemodynamic therapy

Inspired by unique enzyme-mimicking activities of Au and Pt nanocomposites and special roles of Cu components for native and artificial enzymes, we have designed the novel AuPt3Cu trimetallic hetero-structural nanozymes skillfully in this study. AuPt3Cu nanoparticles composed with Au-rich cores and Pt/Cu-rich outer shells with total size of 27.5 ± 3.1 nm are produced in aqueous solutions at 40 °C. Thanks to the alloyed Cu and special hetero structure, AuPt3Cu nanoparticles show excellent oxidase-, peroxidase-, and catalase-like activities. Catalyzed by AuPt3Cu, glucose and glutathione are oxidized by O2 (maximum reaction velocity, vmax, (5.65 ± 0.96) × 10-8, (9.82 ± 1.17) × 10-8 mol·L–1·s−1, respectively) and H2O2 is produced. Further, H2O2 is decomposed into O2 (vmax, 0.06 ± 0.003 mol·L–1·s−1) and reactive oxygen species (ROS, including ·OH, O2·–, 1O2). Then, O2 is decomposed into O2·– radicals. The catalytic reaction velocities of AuPt3Cu are higher than those of AuPt3 and PtCu0.4 counterparts. Because of the cascade reactions in tumor microenvironments, AuPt3Cu nanozymes own remarkable chemodynamic therapy (CDT) performance with IC50 of 50 µg·mL−1 towards cancerous MCF-7 cells. Starving and ROS therapy paths coexist. The tumors of mice bearing with MCF-7 disappeared completely on the 14th day after administering with 5 mg·kg−1 AuPt3Cu agents every 3 days. There are no damages to normal cells and organs, and liver and renal functions. Whether the synthetic strategy or CDT effects, AuPt3Cu nanozymes have unique advantages in cost, performance, and environmental conservation compared to noble metal-, transition metal oxide-, metal complex-, and polymer-relevant drugs.