Kinetic Parameters Research Articles

The effect of gene polymorphism on ticagrelor metabolism: an in vitro study of 22 CYP3A4 variants in Chinese Han population.

Ticagrelor is a novel oral antiplatelet agent which can selectively inhibit P2Y12 receptor. Bleeding and dyspnea are common adverse reactions of ticagrelor in clinic. The side effects of ticagrelor are correlated with the plasma concentration of ticagrelor. This study aimed to evaluate the catalytic characteristics of 22 CYP3A4 alleles identified in the Chinese Han population on the metabolism of ticagrelor in vitro, focusing on the effect of CYP3A4 polymorphism on ticagrelor metabolism. In this study, insect cells were used to express 22 CYP3A4 variants, which were then incubated with 1-50 µM ticagrelor at 37 °C for 30 minutes to obtain the metabolite (AR-C124910XX). AR-C124910XX was detected by UHPLC-MS/MS to calculate the kinetic parameters, including Km, Vmax and CLint. Compared to the wild-type, most CYP3A4 alleles exhibited significant differences in intrinsic clearance. The intrinsic clearance of CYP3A4*11, *18 and *33 was much higher than that of wild-type; four variants exhibited similar intrinsic clearance values as the wild-type enzyme; The remaining 14 variants showed significantly reduced intrinsic clearance values, ranging from 1.48% to 75.11% of the wild-type; CYP3A4*30 displayed weak or no activity. This study conducted a comprehensive assessment of the effect of CYP3A4 variants on ticagrelor's metabolism. The results suggested that there is allele-specific activity towards ticagrelor in vitro. These findings can provide some insights and predictions for treatment strategies and risk assessments associated with ticagrelor in clinical practice.

Identification of a thermostable L-asparaginase from Pyrococcus yayanosii CH1 and its application in the reduction of acrylamide.

L-asparaginase (ASNase, E.C. 3.5.1.1) catalyzes the deamination of L-asparagine to L-aspartic acid and ammonia and is widely used in medicine to treat acute lymphocytic leukemia. It also has significant applications in the food industry by inhibiting acrylamide formation. In this study, we characterized a thermostable ASNase from the hyper thermophilic strain, Pyrococcus yayanosii CH1. The recombinant enzyme (PyASNase) exhibited maximal activity at pH 8.0 and 85°C. Moreover, PyASNase demonstrated promising thermostability across temperatures ranging from 70 to 95°C. The kinetic parameters of PyASNase for L-asparagine were a Km of 6.3mM, a kcat of 1989s-1, and a kcat/Km of 315.7mM-1s-1. Treating potato samples with 10 U/mL of PyASNase at 85°C for merely 10min reduced the acrylamide content in the final product by 82.5%, demonstrating a high efficiency and significant advantage of PyASNase in acrylamide inhibition.

MODELING GROWTH AND VISCOUS FLOW OF OXIDE ON CYLINDRICAL SILICON SURFACES INCLUDING PIEZOVISCOUS INHIBITION

Abstract A baseline constant-parameters non-dimensional cylindrical oxidation model was first revisited, normalizing oxide thickness to oxidant diffusivity/reaction rate ratio instead of original cylinder radius, allowing this radius to remain evident in model output, which in this baseline case predicted oxides increasingly thicker for convex surfaces of decreasing radius as compared to that on flat surface, with oxide thicknesses on concave surfaces instead predicted to decrease with decreasing radius from the flat case. As this convex behavior conflicts with reported experiment, where oxide thickness on convex surfaces rather decrease with decreasing radius from the flat case though less strongly than for concave surfaces, potential stress-dependent kinetics parameters were investigated, with stresses determined from viscous treatment of oxide flow during growth to accommodate its molecular volume exceeding that of the silicon consumed in its production. While oxidant diffusivity and solubility both considered to increase with increasing hydrostatic pressure instead cause model predictions to further deviate from experimental observation, the rate of its volume-producing reaction with the silicon considered to decrease with increasingly compressive radial stress across their interface successfully brought model predictions of convex curvatures into agreement with reported experiments. In conjunction with such stress-dependent reaction rate, additional consideration of viscosity as increasing with hydrostatic pressure can also contribute towards bringing convex modeling closer to experimental observation, though it also introduces a potential piezoviscous inhibition where concave cases of smaller curvature radius become predicted incapable of even initiating oxidation.

Comprehensive thermal properties, kinetic, and thermodynamic analyses of biomass wastes pyrolysis via TGA and Coats-Redfern methodologies

This study comprehensively analyzes the thermal decomposition characteristics as well as the kinetic and thermodynamic parameters of five biomass wastes, including coffee husk, groundnut shell, macadamia nutshell, rice husk, and tea waste, using Thermogravimetric Analysis (TGA) and the Coats-Redfern method. The TGA experiments were conducted on a PerkinElmer STA 6000 instrument under an inert N2 atmosphere with a heating rate of 20 °C/min, spanning a temperature range from 25 °C to 950 °C. The results identified three distinct pyrolysis stages: drying, devolatilization, and char formation, with macadamia nutshell demonstrating the highest thermal reactivity and efficient devolatilization characteristics, reflected by its lowest initial devolatilization temperature (175 °C) and highest peak temperature (380 °C). Kinetic analysis revealed that coffee husk had the highest overall activation energy (Ea) of 60.59 kJ/mol, indicating complex thermal degradation behavior. The thermodynamic evaluation showed that coffee husk also exhibited the highest enthalpy change (ΔH=55.46 kJ/mol) but the lowest Gibbs free energy change (ΔG=148.34 kJ/mol), suggesting high energy requirements for decomposition but relatively more spontaneous reactions compared to other biomass types. Macadamia nutshell demonstrated high ΔG (163.24 kJ/mol) and moderate ΔH (32.44 kJ/mol), reflecting greater resistance to spontaneous decomposition. The comprehensive pyrolysis index (CPI) and devolatilization index (Ddev) confirmed macadamia nutshell as the most reactive biomass, while rice husk exhibited the lowest reactivity. The findings highlight the importance of multi-step kinetic analysis for accurately understanding pyrolysis processes, providing critical insights for optimizing biomass conversion for energy production. Future research should explore co-pyrolysis with varied biomass mixtures and advanced kinetic modeling to enhance energy yields.

Kinetic parameters of thermal decomposition of biofuels and its oil-containing composites

The kinetic features of the decomposition of fuel pellets formed from birch phloem and its composites with the addition of oil-containing waste (OCW) were studied by methods of thermogravimetric analysis carried out in various atmospheric conditions. The characteristic temperature ranges of thermal decomposition of the materials from which pellets are formed were identified, the rates of mass loss and activation energy at the main stages of thermal decomposition were evaluated, and the combustion indices of composite compositions were determined to identify the possibility of using such compositions as alternative fuels. By evaluation of activation energies for each stage of sample decomposition, the limiting influence of certain stages on the process of thermal destruction was established.

Isothermal co‐pyrolytic kinetics investigation of polystyrene/polymethyl methacrylate blended Bakelite

AbstractThe widespread use of Bakelite, polystyrene (PS), and polymethylmethacralate (PMMA) has caused significant pollution, requiring advanced recycling methods. Pyrolysis, co‐pyrolysis, and catalytic co‐pyrolysis are key for recycling these wastes, necessitating kinetic studies and specific reactor designs of their thermal degradation. Isothermal thermogravimetric analysis at 300, 350, 400, 450, and 500°C was used to study thermal degradation kinetics, based on the non‐isothermal degradation zone of Bakelite. The batch pyrolysis of discarded Bakelite and PS/PMMA–Bakelite blends was conducted at 450°C. The thermal decomposition of Bakelite and its blends increases with higher isothermal pyrolytic temperatures. The addition of PS or PMMA to Bakelite substantially accelerates its thermal decomposition. The maximum weight loss of Bakelite, PS–Bakelite, and PMMA–Bakelite are 55%, 96.75%, and 89.51% at 500°C, respectively. The kinetic analysis is crucial for designing specific reactors, utilizing the D1‐diffusion‐based method for Bakelite, with an activation energy (Ea) of 17.178 kJ/mol and Arrhenius constant (A) of 0.095 min−1. The A2‐ and A3‐Avrami–Erofeyev methods explain the isothermal degradation of PS–Bakelite and PMMA–Bakelite blends, with activation energies of 9.031 and 12.59 kJ/mol, and Arrhenius constants of 0.056 and 0.075 min−1, respectively. The co‐pyrolysis of PS–Bakelite yields the highest condensable products (66.76%) and needs the longest reaction time (320 min). The Fourier transform Infrared (FTIR) and gas chromatography–mass spectrometry (GC–MS) analyses confirm the presence of alkanes, cycloalkanes, alkenes, cycloalkenes, aromatic hydrocarbons, and oxygenated compounds in the pyrolytic oils. This study provides unique kinetic parameters and product analyses, showing effects of blending on the decomposition rates and yields valuable compounds, advancing recycling technologies.

Experimental and kinetics of the oxidation of ammonium sulfite by hydrogen peroxide in ammonia flue gas desulfurization

ABSTRACT The oxidation of ammonium sulfite obtained from ammonia-based flue gas desulfurization by hydrogen peroxide in aqueous solutions was studied using a stirred reactor. An experimental study of the kinetic parameters of the oxidation process was investigated in the pH of 4.2–5.2 at 25–50°C. r = k 3 H + HS O 3 − H 2 O 2 is valid at sulfite concentrations of 10−4 mol/L for the pH range 4.2–5.2. At 298K and pH of 5.0, third-order rate constant k 3 was found to be (6.9 ± 0.21)×107 (mol/L)−2s−1. When the concentration of sulfite is 0.01 mol/L, the reaction first-order with respect to sulfite and hydrogen peroxide, and the second-order reaction rate constant at pH of 4.2–5.4 is lg k 2 = − 1.0135 pH + 7.4862 . Activation energy of E a = 32.26 kJ/moL. Kinetic data were compared with previous studies. A semi-empirical formula can express the effect of ionic strength on k 3. The kinetic data of ammonium sulfite oxidation with hydrogen peroxide have potential applications in the wet ammonia desulfurization process.

An in vitro set-up to study Pdr5-mediated substrate translocation.

Pdr5 is the most abundant ABC transporter in Saccharomyces cerevisiae and plays a major role in the pleiotropic drug resistance (PDR) network, which actively prevents cell entry of a large number of structurally unrelated compounds. Due to a high level of asymmetry in one of its nucleotide binding sites (NBS), Pdr5 serves as a perfect model system for asymmetric ABC transporter such as its medical relevant homologue Cdr1 from Candida albicans. In the past 30 years, this ABC transporter was intensively studied in vivo and in plasma membrane vesicles. Nevertheless, these studies were limited since it was not possible to isolate and reconstitute Pdr5 in a synthetic membrane system while maintaining its activity. Here, the functional reconstitution of Pdr5 in a native-like environment in an almost unidirectional inside-out orientation is described. We demonstrate that reconstituted Pdr5 is capable of translocating short-chain fluorescent NBD lipids from the outer to the inner leaflet of the proteoliposomes. Moreover, this transporter revealed its ability to utilize other nucleotides to accomplish transport of substrates in a reconstituted system. Besides, we were also able to estimate the NTPase activity of reconstituted Pdr5 and determine the kinetic parameters for ATP, GTP, CTP, and UTP.

Microbial community respiration kinetics and their dynamics in coastal seawater

Oxygen (O2) concentrations in coastal seawater have been declining for decades and models predict continued deoxygenation into the future. As O2 declines, metabolic energy use is progressively channelled from higher trophic levels into microbial community respiration, which in turn influences coastal ecology and biogeochemistry. Despite its critical role in deoxygenation and ecosystem functioning, the kinetics of microbial respiration at low O2 concentrations in coastal seawater remain uncertain and are mostly modeled based on parameters derived from laboratory cultures and a limited number of environmental observations. To explore microbial responses to declining O2, we measured respiration kinetics in coastal microbial communities in Hong Kong over the course of an entire year. We found the mean maximum respiration rate (Vmax) ranged between 560 ± 280 and 5930 ± 800 nmol O2 L−1 h−1, with apparent half-saturation constants (Km) for O2 uptake of between 50 ± 40 and 310 ± 260 nmol O2 L−1. These kinetic parameters vary seasonally in association with shifts in microbial community composition that were linked to nutrient availability, temperature, and biological productivity. In general, coastal communities in Hong Kong exhibited low affinities for O2, yet communities in the dry season had higher affinities, which may play a key role in shaping the relationship between community size, biomass, and O2 consumption rates through respiration. Overall, parameters derived from these experiments can be employed in models to predict the expansion of deoxygenated waters and associated effects on coastal ecology and biogeochemistry.

Molecular Identification and Engineering a Salt-Tolerant GH11 Xylanase for Efficient Xylooligosaccharides Production.

This study identified a salt-tolerant GH11 xylanase, Xynst, which was isolated from a soil bacterium Bacillus sp. SC1 and can resist as high as 4 M NaCl. After rational design and high-throughput screening of site-directed mutant libraries, a double mutant W6F/Q7H with a 244% increase in catalytic activity and a 10 °C increment in optimal temperature was obtained. Both Xynst and W6F/Q7H xylanases were stimulated by high concentrations of salts. In particular, the activity of W6F/Q7H was more than eight times that of Xynst in the presence of 2 M NaCl at 65 °C. Kinetic parameters indicated they have the highest affinity for beechwood xylan (Km = 0.30 mg mL-1 for Xynst and 0.18 mg mL-1 for W6F/Q7H), and W6F/Q7H has very high catalytic efficiency (Kcat/Km = 15483.33 mL mg-1 s-1). Molecular dynamic simulation suggested that W6F/Q7H has a more compact overall structure, improved rigidity of the active pocket edge, and a flexible upper-end alpha helix. Hydrolysis of different xylans by W6F/Q7H released more xylooligosaccharides and yielded higher proportions of xylobiose and xylotriose than Xynst did. The conversion efficiencies of Xynst and W6F/Q7H on all tested xylans exceeded 20%, suggesting potential applications in the agricultural and food industries.

Exploration of modified β-cyclodextrin compounds as enhanced eco-friendly and easily accessible corrosion inhibitors for carbon steel in hydrochloric acid

β-CD was esterified independently with two distinct ionic liquids to evaluate their potential as corrosion inhibitors for carbon steel in a 1.0 M HCl solution. Experimental and computational methods were used to assess the inhibitors’ efficiency regarding temperature and concentration effects and to elucidate the adsorption mechanism. Both inhibitors acted as barriers, with a maximum inhibition efficiency of about 94 % at 200 ppm. There was a direct relationship between the inhibitor's concentration and their efficiency, while increasing the temperature reduced the inhibitors’ effectiveness. The thermodynamic and kinetic parameters were evaluated and studied. DFT calculation revealed the electronic structure and reactivity of the inhibitors. Molecular dynamic simulation provided insight into the optimal configuration and binding energies of inhibitors on the Fe (110) surface.

Facile oxygen-enriched hierarchical porous carbon particles based on biomass wastes for improving the adsorption of malachite green from aqueous solutions

The abundance of active groups and the surface areas of adsorbents used for the removal of contaminants such as dyes are very important for the effective adsorption process. For this purpose, the high surface area-activated carbon was obtained by activation of agricultural waste chestnut shells as carbon precursor(CSAC) with sodium hydroxide in the first stage of this study. In the second stage, the doping of oxygen atoms with nitric acid was carried out to further improve the surface properties of this activated carbon (O-doped CSAC). The obtained oxygen-rich activated carbon sample was used as an adsorbent to remove a cationic dye malachite green (MG). EDS, SEM, XRD, FTIR and nitrogen adsorption analyses were used for the characterisation of these activated carbon samples. FTIR, elemental analysis and EDS analyses showed a significant increase in the amount of oxygen groups on the surface. The BET surface area obtained for CSAC had a high surface area of 1548 m2/g. Kinetic, thermodynamic and isotherm parameters for the adsorption of MG with O-doped CSAC were determined. The qe values obtained for MG adsorption with O-doped CSAC showed an adsorption efficiency of about 90 %. An adsorption capacity of 284.6 mg g-1 was obtained at 298 K with the Langmuir isotherm. The adsorption mechanism of MG with O-doped CSAC was also investigated.

Growth of Lactiplantibacillus plantarum BG112 in Batch and Continuous Culture with Camellia sinensis as Prebiotic

This work aimed to study the effect of Camellia sinensis extract (CSExt) as a particular growth promoter of Lactiplantibacillus plantarum (LP) in batch and continuous production processes. Growth conditions were 1% (v/v) inoculum, pHC = 6.5, 1% of dissolved oxygen (D.O.), 37 °C, and 150 rpm in a 0.2 L bioreactor using a commercial MRS broth (de Man, Rogosa, and Sharpe) and 1% (v/v) or 10% (v/v) CSExt according to the experimental design. In batch experiments, the maximum specific growth rate and the affinity constant increased with the increase in CSExt. In continuous culture, biomass production increased significantly with the addition of 1% (w/v) CSExt at 0.15 (1/h). Kinetic parameters adjusted were similar to those reported in the literature. Substrate affinity and the specific growth rate increased significantly in the presence of CSExt in batch and continuous cultures. Based on the results, prebiotics from plant extracts may function as growth promoters in specific physiological stages. This is the first report showing the change in kinetic parameters of a probiotic strain growing in crude plant extracts.

Differential roles of kinetic on- and off-rates in T-cell receptor signal integration revealed with a modified Fab’-DNA ligand

Antibody-derived T-cell receptor (TCR) agonists are commonly used to activate T cells. While antibodies can trigger TCRs regardless of clonotype, they bypass native T cell signal integration mechanisms that rely on monovalent, membrane-associated, and relatively weakly binding ligand in the context of cellular adhesion. Commonly used antibodies and their derivatives bind much more strongly than native peptide major histocompatibility complex (pMHC) ligands bind their cognate TCRs. Because ligand dwell time is a critical parameter that tightly correlates with physiological function of the TCR signaling system, there is a general need, both in research and therapeutics, for universal TCR ligands with controlled kinetic binding parameters. To this end, we have introduced point mutations into recombinantly expressed α-TCRβ H57 Fab to modulate the dwell time of monovalent Fab binding to TCR. When tethered to a supported lipid bilayer via DNA complementation, these monovalent Fab'-DNA ligands activate T cells with potencies well-correlated with their TCR binding dwell time. Single-molecule tracking studies in live T cells reveal that individual binding events between Fab'-DNA ligands and TCRs elicit local signaling responses closely resembling native pMHC. The unique combination of high on- and off-rates of the H57 R97L mutant enables direct observations of cooperative interplay between ligand binding and TCR-proximal condensation of the linker for activation of T cells, which is not readily visualized with pMHC. This work provides insights into how T cells integrate kinetic information from TCR ligands and introduces a method to develop affinity panels for polyclonal T cells, such as cells from a human patient.

An intriguing way to synthesize a TiO2–ZnO hybrid nanostructure for the pragmatic application as a photocatalyst

In an attempt to counter the problem of treating wastewater more efficiently, we report the study on photocatalytic properties of metal oxide nanostructures under different light sources. A novel sonication-assisted refluxing method was employed to synthesize a binary heterostructure consisting of TiO2 and ZnO. XRD and EDS analysis of synthesized materials confirm the presence of both TiO2 and ZnO. Also, the change in the intensity of XRD peaks and elemental composition are well observed with a change in molar concentration of Ti and Zn. HR-TEM micrographs show nanostructures having hybrid Janus and Core-shell type structures. The XPS spectra were probed to identify the surface chemical species associated with the prepared heterostructures. Raman spectra confirm the smaller ZnO particles attached over agglomerated TiO2 as a shell layer as depicted in TEM results. Optical studies exhibit the modification in the band structure of the materials due to the formation of the heterostructure. The synthesized hybrid catalyst shows impressive results in photocatalytic performance. The catalytic potency of the prepared heterostructures under UV light is reminiscent of Anatase while it gets convalescent with an increment of Zn content under visible light. The photocatalytic performance of synthesized materials under sunlight shows their potential to be utilized as commercial catalysts for direct application in wastewater treatment. The kinetic study of the degradation data reveals interesting facets of the catalysis. It suggests that the photo-corrosion of the catalyst inordinately changes the rate of photocatalytic reaction which ultimately affects the photocatalytic efficiency of the catalyst. The study also discusses the role of kinetic and validation parameters in the selection of the best-fitted kinetic model with realistic arguments.

The oral intake of specific Bioactive Collagen Peptides (BCP) improves gait and quality of life in canine osteoarthritis patients—A translational large animal model for a nutritional therapy option

Objective Osteoarthritis (OA) is the most common joint disorder in humans and dogs. Due to its chronic progressive nature, the predominant clinical signs after a certain point are pain and immobility. The similar pathogenesis allows conclusions to be drawn from canine to human OA. Current treatments are limited and often attempt to treat OA symptoms rather than improve joint structure and function. Collagen hydrolysates as oral supplements are a promising therapeutic option to achieve this advanced therapeutic aim in both species. The effects of oral supplementation were therefore investigated in canine OA patients. Method In a systematic, placebo-controlled, double-blind interventional study in 31 dogs with naturally occurring OA, the efficacy of oral supplementation of specific bioactive collagen peptides (BCP) was tested in comparison to the approved combination of the active substances omega-3 fatty acids and vitamin E. The dogs were examined on a horizontal treadmill with 4 integrated piezoelectric force plates at the beginning and end of a twelve-week test period. At both points, the owners completed a specific questionnaire containing the validated Canine Brief Pain Inventory (CBPI) and the dogs were fitted with accelerometers to record total daily activity data. Results Only the oral supplementation of BCP resulted in a significant improvement of several kinetic parameters measured using a force-plate fitted treadmill, and the quality of life assessed by CBPI, while accelerometry was unaffected by the intervention. Conclusion The results of this three-month BCP supplementation study using objective measurement parameters in dogs with naturally occurring OA demonstrate an efficacy, suggesting the therapeutic use of BCP in canine OA patients and demonstrating the relevance of this collagen hydrolysate formulation for the treatment of OA in human patients as well.

Debromination of Pyrolytic oil from waste printed circuit boards by catalytic thermo chemical reactions with Ca(OH)2 and ZSM-5

The surge in Waste Electrical and Electronic Equipment (WEEE) volume presents a formidable disposal challenge. This study focused on catalytic pyrolysis of waste printed circuit boards (WPCBs) employing Ca(OH)2 alone and in conjunction with ZSM-5 as catalysts. Kinetic parameters for catalytic and non-catalytic pyrolysis were derived through thermogravimetric analysis (TGA). The Coats-Redfern method for non-catalytic pyrolysis showcased activation energies of 33.52, 405.81, and 67.96 kJ/mol in zones 1, 2, and 3, respectively with corresponding reaction orders of 0.9, 2.8, and 1.2. Introduction of Ca(OH)2 amplified activation energy and reaction order in zones 2 and 3. Subsequent incorporation of ZSM-5 with Ca(OH)2 resulted in reduced activation energy and reaction order in zone 2, while elevating them in zones 1 and 3. Validation through laboratory-scale fixed-bed reactor experiments confirmed TGA findings and unveiled that pyrolysis oil had enhanced phenolic yield because of ZSM-5 where Ca(OH)2 showcased its effectiveness in eliminating halogens.

Multi‐response kinetic study of Maillard reaction hazards in the glucose‐lysine model system

AbstractBACKGROUNDNε‐carboxymethyllysine (CML), Nε‐carboxyethyllysine (CEL) and α‐aminoadipic acid (AAA) are important foodborne hazards and their intake can cause a variety of diseases in humans. It is extremely important to investigate the formation mechanism of CML, CEL and AAA, as well as their association with each other when aiming to control their production.RESULTSA multi‐response kinetic model was developed within the glucose‐lysine Maillard reaction model system. The concentrations of glucose, lysine, glyoxal (GO), methylglyoxal (MGO), CML, CEL and AAA were quantified at different temperature (100–160 °C) and at different intervals (0–60 min). The experimental data were fitted to the proposed model to calculate kinetic parameters for the corresponding steps. The results indicated that the production of CML was primarily relied on the direct oxidative cleavage of the Amadori product, rather than the reaction between GO and Lys, whereas CEL and AAA were generated through the reaction of MGO with Lys. Significantly, the reaction between α‐dicarbonyl compounds and Lys preferentially generated CML and CEL, resulting in the lower concentrations of AAA compared to CML and CEL.CONCLUSIONThe multi‐response kinetic model developed in the present study can be applied well to the Maillard reaction. The relationship between the formation mechanisms of CML, CEL and AAA is also explained. © 2024 Society of Chemical Industry.

Biochemical and toxicological characteristics of polyphenol oxidase from red palm weevil Rhynchophorus ferrugineus (Olivier) (Coleoptera: Curculionidae)

Red palm weevil (RPW) Rhynchophorus ferrugineus is the most destructive insect pests of numerous palm species in the world. The introduction of botanical extract(s) as integral part of an integrated pest management (IPM) programs against RPW will reduce the use of chemical insecticides. Polyphenol oxidase (PPO) is one of the RPW innate immune mechanisms and inhibition of such enzyme could result in a disorder of the insect's immune system. A one single PO isoenzyme has been purified from the hemolymph of the 12th instar larvae of RPW. Using L-DOPA as substrate, R. ferrugineus PPO exhibited specific activity 428 Units/mg proteins with 8.3-fold purification, optimum pH and temperature for activity at 7.5 and 40 °C, respectively and is enhanced by Cu2+ with 1.76-fold. The rank order for oxidizing R. ferrugineus PPO different substrates is catechol > pyrogallol > L-DOPA > pyrocatechuic acid and not tyrosine. The kinetic parameters Km, Vmax and Vmax/Km for L-DOPA are 3.3 mM, 1.3 μmol/ml/min, and 0.39, respectively. The catalytic efficiency of the enzyme towards catechol is 5.3-fold higher than that for L-DOPA. The enzyme completely inhibited by thiourea, ascorbic acid, dithiothreitol, and SDS. R. ferrugineus PPO is a catechol oxidase di-phenol: O2 oxidoreductase. Based on the toxicological studies of various botanical extracts, the IC50 ranged from 20 to 90 mg/ml. The enzyme completely inhibited by 50 mg/ml Cinnamomum camphora. Gallic acid, the major phenolic compound, has IC50 0.8 mM and competitively inhibited the enzyme with Ki 0.54 mM. C. camphora could be a useful natural RPW-controlling agent and used as integral part in IPM programs. This interpretation can be validated in future through an in vivo investigation.

Design and synthesis of spiro[pyrrolidine-3,3′-quinoline]-2,2′-dione derivatives as novel antifungal agents targeting chitin synthase

A novel spiro [pyrrolidine-3′,3′-quinoline]-2,2′-dione scaffold was constructed using fragments of quinoline and pyrrolidine. Subsequently, two series of derivatives were designed based on this scaffold. The enzyme inhibition experiments revealed that all designed compounds had moderate to good inhibitory activity against chitin synthase (CHS). The inhibitory effects of compounds 5i, 5j, 8i and 8n were approximately equal to that of control drug polyoxin B (PB, IP = 86.4 ± 2.9 %, IC50 = 0.082 ± 0.013 mM) which is a well-established CHS inhibitor. The results from enzyme kinetic parameters assays proved that these compounds act as non-competitive inhibitors of CHS. The sorbitol protection experiments demonstrated the tested compounds disrupted the synthesis of cell wall, which further verified that the target of these compounds is CHS. The experiments of antimicrobial showed that compounds 5b, 5f, 5i, 5j, 8f, 8i, 8m, 8n and 8o had strong antifungal activity against the four tested pathogenic fungi strains frequently emerging in clinical setting, with MIC values ranging from 4 to 32 μg/mL, which were either superior to or comparable with those of PB or fluconazole. Furthermore, these compounds displayed synergistic or additive effects when combined with fluconazole and these active compounds also showed promising activity against fluconazole-resistant and micafungin-resistant fungi variants. The result of antimicrobial experiments indicated that these compounds had minimal activity to tested bacterial strains. This suggests that they had selective antifungal activity. The results of ADME prediction, in conjunction with the cytotoxicity assay results, indicated that these compounds had favorable pharmacokinetic profiles and low toxicity. In addition, molecular docking studies illustrated that the compound had a strong affinity with the CHS, which was consistent with the results of enzymatic assays. These findings indicated that the designed compounds are non-competitive inhibitors of CHS with good selectivity and broad-spectrum antifungal activity, and possess significant antifungal activity against drug-resistant fungi, suggesting their potential as lead compounds for the development of novel drugs against drug-resistant fungi.