Kinetic Parameters Research Articles

Immobilization of Xylanase on ZnO nanoparticles obtained by green synthesis from Eupatorium cannabinum L. and its application in enrichment of fruit juices

Juice production is one of the main components of the nutritional food industry. Therefore, improving fruit juice quality and preventing the formation of negative by-products is important for human health. The role of environmentally friendly, safe and economically more affordable green nanocarriers in improving the quality of fruit juice is increasing day by day. For this purpose, zinc oxide nanoparticles (ZnONPs) were prepared from the aqueous extract of the Eupatorium cannabinum L. using the green synthesis method. After immobilization, structural and morphological characterizations of the prepared ZnONPs and xylanase (Xyl)@ZnONPs were carried out, which confirmed that both the synthesis of ZnONPs and the immobilization of Xyl enzyme on ZnONPs were successfully carried out. The biochemical parameters of Xyl@ZnONPs such as optimum pH and temperature, kinetic parameters, thermal stability, reusability, and storage stability were investigated and compared with free Xyl. After immobilization, the optimum pH of free Xyl shifted from 5.0 to an alkaline pH of 8.0, while no change in the optimum temperature (70 °C) was observed for both enzymes. While the activation energy of free Xyl was 5.56 kJ/mol, it was found to be 7.27 kJ/mol for Xyl@ZnONPs. Xyl@ZnONPs outperformed free Xyl due to its long-term storage stability and reusability. Moreover, Xyl@ZnONPs retained 50 % of its activity after 5 reuses and 87 % of its activity after 4 weeks of storage at room temperature. In addition, a decrease in Km value and an increase in Vmax value were observed after immobilization, which means the increased catalytic activity of the Xyl@ZnONPs. As a result of re-using Xyl@ZnONPs in orange juice for 6 cycles, clarification yield of 66.43 % was obtained under optimized conditions. Overall, this study prepared a suitable, environmentally friendly, and robust carrier support for Xyl immobilization and developed a biocatalyst that will provide high performance in the industrial field.

Michaelis-like complex of mouse ketohexokinase isoform C.

Over the past forty years there has been a drastic increase in fructose-related diseases, including obesity, heart disease and diabetes. Ketohexokinase (KHK), the first enzyme in the liver fructolysis pathway, catalyzes the ATP-dependent phosphorylation of fructose to fructose 1-phosphate. Understanding the role of KHK in disease-related processes is crucial for the management and prevention of this growing epidemic. Molecular insight into the structure-function relationship in ligand binding and catalysis by KHK is needed for the design of therapeutic inhibitory ligands. Ketohexokinase has two isoforms: ketohexokinase A (KHK-A) is produced ubiquitously at low levels, whereas ketohexokinase C (KHK-C) is found at much higher levels, specifically in the liver, kidneys and intestines. Structures of the unliganded and liganded human isoforms KHK-A and KHK-C are known, as well as structures of unliganded and inhibitor-bound mouse KHK-C (mKHK-C), which shares 90% sequence identity with human KHK-C. Here, a high-resolution X-ray crystal structure of mKHK-C refined to 1.79 Å resolution is presented. The structure was determined in a complex with both the substrate fructose and the product of catalysis, ADP, providing a view of the Michaelis-like complex of the mouse ortholog. Comparison to unliganded structures suggests that KHK undergoes a conformational change upon binding of substrates that places the enzyme in a catalytically competent form in which the β-sheet domain from one subunit rotates by 16.2°, acting as a lid for the opposing active site. Similar kinetic parameters were calculated for the mouse and human enzymes and indicate that mice may be a suitable animal model for the study of fructose-related diseases. Knowledge of the similarity between the mouse and human enzymes is important for understanding preclinical efforts towards targeting this enzyme, and this ground-state, Michaelis-like complex suggests that a conformational change plays a role in the catalytic function of KHK-C.

Environmentally safe chemometric evaluation and data analysis for the kinetic investigation of mirabegron stability

The physicochemical characteristics of different pharmaceuticals may change with transportation, storage, and usage which directly affect their efficacy, quality, and other features. It is crucial to investigate the kinetic action of pharmaceutical compounds to produce predictive data that aid in the computation of their shelf lives. Several conventional analytical methods introduce unwanted dangerous risks, like employing unsafe solvents, that harm both the analyst and the environment. This created the motive for analysts to apply green approaches that consider using safe chemicals and producing as little waste as possible. In this study, powerful green assessment methods were investigated to study the kinetic process of Mirabegron (MIR) in different hydrolytic conditions giving its main degradation product. First, the optimal solvent was identified for the given kinetic procedures with the aid of Principal Component Analysis. Then two advanced chemometric methods were applied to estimate the kinetic parameters of the MIR degradation process namely: Multivariate Curve Resolution-Alternating Least Squares and Artificial Neural Networks. By data manipulation, both algorithms could provide an explanation of the MIR kinetic process in different hydrolytic conditions. Moreover, modified National Environmental Methods Index (NEMI), eco-scale, Hexagon algorithm, Complementary Green Analytical Procedure Index (Complex GAPI), and analytical greenness metric for sample preparation (AGREEprep) were adopted to ensure the greenness of the proposed study. Eventually, the presented investigation could successfully present an eco-friendly chemometric approach for monitoring the different kinetic processes of pharmaceutical compounds with minimum effort and solvent consumption and maximum time saving and analyst safety compared to other traditional methods.

Characterization of the interaction between the Sec61 translocon complex and ppαF using optical tweezers.

The Sec61 translocon allows the translocation of secretory preproteins from the cytosol to the endoplasmic reticulum lumen during polypeptide biosynthesis. These proteins possess an N-terminal signal peptide (SP) which docks at the translocon. SP mutations can abolish translocation and cause diseases, suggesting an essential role for this SP/Sec61 interaction. However, a detailed biophysical characterization of this binding is still missing. Here, optical tweezers force spectroscopy was used to characterize the kinetic parameters of the dissociation process between Sec61 and the SP of prepro-alpha-factor. The unbinding parameters including off-rate constant and distance to the transition state were obtained by fitting rupture force data to Dudko-Hummer-Szabo models. Interestingly, the translocation inhibitor mycolactone increases the off-rate and accelerates the SP/Sec61 dissociation, while also weakening the interaction. Whereas the translocation deficient mutant containing a single point mutation in the SP abolished the specificity of the SP/Sec61 binding, resulting in an unstable interaction. In conclusion, we characterize quantitatively the dissociation process between the signal peptide and the translocon, and how the unbinding parameters are modified by a translocation inhibitor.

Physical and Numerical Modeling of Flow in a Meandering Channel

In this study, flow behavior in a meandering channel was investigated experimentally and numerically. The experiments were carried out for nine different cases on a channel consisting of 180° and 120° angle bends following successively. Measurements were conducted employing Acoustic Doppler Velocimetry (ADV) at 13 different points in the inner, middle, and outer bends of the sections located at significant bends along the channel. Depth-averaged velocity, velocity profiles, bed shear stress, and turbulence kinetic energy parameters were considered to understand the flow behavior in the meandering channel. A 1:1 scale numerical model of the experimental setup was generated using the Computational Fluid Dynamics (CFD) method through the verified FLOW-3D software (HYDRO 2022R1). It was found to be successful in estimating all parameters and was capable of investigating the flow behavior in the meandering channel. Additionally, a mesh independence study was performed, and four different turbulence models were compared. As a result, as the flow encountered the first meander in the channel, secondary flow occurred, and lateral momentum transfer took place. Therefore, velocity increased by approximately 30% from the first meander of 180° angles to the second meander of 120° angles. Therefore, the most critical zone was the inner bend of the 120-angle meander.

In vitro, in situ, and in vivo evaluation of a sesame paste supplement for grazing calves

Objective: To carry out an in vitro, in situ, and in vivo evaluation of soybean paste (T1) and sesame paste (T2) supplements for calves under grazing conditions. Methodology: Partial (24, 48, and 72 h) and accumulated biogas and methane production, as well as dry matter degradation (DMD), were measured in the in vitro analysis. The digestibility kinetics parameters (a, b, a+b, c, k, ED) were estimated in the in situ test. Feed supplement consumption (SC), daily weight gain (DWG), bacterial and protozoan populations, acetate, propionate, and butyrate were evaluated in the in vivo test. A completely randomized experimental design was used. Results: T1 produced a higher amount of partial and accumulated biogas and methane at 24 h, as well as the highest values in the b, a+b, k, and ED (p ≤ 0.05) parameters. Meanwhile the rest of the in vitro, in situ, and in vivo variables did not record any differences between treatments (p > 0.05). Study Limitations/Implications: The production of sesame paste is seasonal; consequently, its harvest limits its year-round availability. Conclusions: Based on the in vitro, in situ, and in vivo evaluations, sesame paste is a feasible option to replace soybean paste as a source of protein, in the supplementation of calves in the tropics.

Molecular Mechanisms Responsible KPC-135-Mediated Resistance to Ceftazidime-Avibactam in ST11-K47 Hypervirulent Klebsiella pneumoniae

Ceftazidime-avibactam resistance attributable to the bla KPC-2 gene mutation is increasingly documented in clinical settings. In this study, we characterized the mechanisms leading to the development of ceftazidime-avibactam resistance in ST11-K47 hypervirulent Klebsiella pneumoniae that harbored the bla KPC-135 gene. This strain possessed fimbriae and biofilm, demonstrating pathogenicity. Compared with the wild-type KPC-2 carbapenemase, the novel KPC-135 enzyme exhibited a deletion of Glu168 and Leu169 and a 15-amino acid tandem repeat between Val262 and Ala276. The bla KPC-135 gene was located within the Tn6296 transposon truncated by IS26 and carried on an IncFII/IncR-type plasmid. Compared to the bla KPC-2-positive cloned strain, only the MIC of ceftazidime increased against bla KPC-135-positive K. pneumoniae and wasn’t inhibited by avibactam (MIC 32 μg/mL), while clavulanic acid and vaborbactam demonstrated some inhibition. Kinetic parameters revealed that KPC-135 exhibited a lower Km and kcat/Km with ceftazidime and carbapenems, and a higher (∼26-fold) 50% inhibitory concentration with avibactam compared to KPC-2. The KPC-135 enzyme exerted a detrimental effect on fitness relative to the wild-type strain. Furthermore, this strain possessed hypervirulent determinants, which included the IncHI1B/FIB plasmid with rmpA2 and expression of type 1 and 3 fimbriae. In conclusion, we reported a novel KPC variant, KPC-135, in a clinical ST11-K47 hypervirulent K. pneumoniae strain, which conferred ceftazidime-avibactam resistance, possibly through increased ceftazidime affinity and decreased avibactam susceptibility. This strain simultaneously harbored resistance and virulence genes, posing an elevated challenge in clinical treatment.

SBMLToolkit.jl: a Julia package for importing SBML into the SciML ecosystem.

Julia is a general purpose programming language that was designed for simplifying and accelerating numerical analysis and computational science. In particular the Scientific Machine Learning (SciML) ecosystem of Julia packages includes frameworks for high-performance symbolic-numeric computations. It allows users to automatically enhance high-level descriptions of their models with symbolic preprocessing and automatic sparsification and parallelization of computations. This enables performant solution of differential equations, efficient parameter estimation and methodologies for automated model discovery with neural differential equations and sparse identification of nonlinear dynamics. To give the systems biology community easy access to SciML, we developed SBMLToolkit.jl. SBMLToolkit.jl imports dynamic SBML models into the SciML ecosystem to accelerate model simulation and fitting of kinetic parameters. By providing computational systems biologists with easy access to the open-source Julia ecosystevnm, we hope to catalyze the development of further Julia tools in this domain and the growth of the Julia bioscience community. SBMLToolkit.jl is freely available under the MIT license. The source code is available at https://github.com/SciML/SBMLToolkit.jl.

Determination of phase transformation kinetics under magnetic fields: Modeling based on magnetization and application in a Fe-1 wt.%Cu alloy

A phase transformation model based on magnetization is proposed in this paper, which accurately tracks the change in the phase transformation volume fraction with time/temperature f-T/t by analyzing phase transformation magnetization curves under a magnetic field. This allows for the determination of kinetic parameters related to the nucleation and growth processes such as the phase transformation rate df/dt-T/t and Avrami exponent n, enabling quantitative analysis of phase transformation kinetics under magnetic field effects. Additionally, the phase transformation magnetization under a magnetic field can be accurately fitted by combining the volume fraction calculation model with the Johnson–Mehl–Avrami equation, thus also obtaining the kinetics parameters. The aforementioned two models are applied to study the isothermal and isokinetic transformations of austenite (γ) to ferrite (α) in Fe-1 wt. %Cu alloys, demonstrating the effects of external conditions through variations in kinetic parameters.

Anaerobic digestion of spoiled milk from dairy industry for biogas production – optimization of operating parameters and kinetic modeling of the pilot scale study

BackgroundSpoiled milk from the dairy industry was subjected to anaerobic treatment to produce biogas at 37℃ in this experiment. Parameters such as inoculum dosage, pH, Chemical Oxygen Demand (COD), and retention time were optimized in a laboratory-scale batch reactor for 90 days.MethodsThe anaerobic digestion of spoilt milk was carried out in a laboratory setting using a batch reactor. Then, using the recognized protocols of the APHA, the characteristics of the spoilt milk were assessed. In order to enhance the accuracy of predicting the reactor's performance, the research adopted two different models for kinetic analysis: the Stover-Kincannon model and the Grau second-order multi-component model. The reactor's improved performance, as indicated by evaluated kinetic parameters, was shown by the superior results from both of these models.ResultsThe results attained from the reactor’s performance were then used as a reference to improve biogas production in a 100 L Anaerobic Sequential Batch Reactor (ASBR) for 45 days. The ASBR achieved a high COD removal efficiency of 92.4% and produced a maximum of 70.4 L of biogas per liter of spoiled milk, equivalent to 69.6% methane content.ConclusionThe Stover-Kincannon model yielded kinetic parameters of Umax = 0.295 gCOD/L and KB = 12.87 gCOD/L, whereas the Grau second-order model presented kinetic coefficients a = 6.744 and b = 2.578. The results obtained from the two models suggest that the investigated kinetic coefficients could be improved upon to increase the reactor's capability for handling different substrates during the AD process.

Thermogravimetric pyrolysis investigation of pistachio shell for its potential of thermal properties, kinetics and thermodynamics

In order to design and optimize thermochemical systems for the production of bioenergy understanding the energy capacity of the pistachio shell and its kinetics of degradation is crucial. The Thermogravimetric analysis at different heating rates of biomass was studied along with thermodynamic study, primary characterizations like the composition of biomass, heating value and a reaction mechanism-assisted kinetic model. Nitrogen gas flow rate is used for thermogravimetric analysis (TGA), which is performed at 10, 15, 20 and 30 ℃/min different heating rates from room temperature to 900 ℃. The output also shows that maximal degradation took place between 180 and 430 °C. The various iso-conversional models, such as Flynn–Wall–Ozawa (FWO), Kissinger–Akahira–Sunose (KAS), Starink and Friedman, are used to calculate kinetic and thermodynamic parameters, which give the average value of activation energy as 172.29, 170.47, 169.79, and 193.25 kJ/mol. The average values of Gibbs free energy for the FWO, KAS, Starink and Friedman techniques were found to be 176.49, 1176.54, 176.29, and 175.78 kJ/mol, respectively.Graphical

Thermophilic-mesophilic temperature phase anaerobic co-digestion compared with single phase co-digestion of sewage sludge and food waste

Anaerobic co-digestion is an effective method for addressing the issue of a single substrate not being able to achieve optimal conditions for anaerobic digestion. By adjusting the mixture ratio of sewage sludge and food waste to achieve the optimal carbon to nitrogen ratio, the effectiveness of thermophilic–mesophilic temperature phase anaerobic co-digestion (TPAcD) was evaluated in comparison to single phase mesophilic anaerobic co-digestion (MAcD) and thermophilic anaerobic co-digestion (TAcD). The results indicated that TPAcD increased methane yield by 50.3% and 32.7% compared to MAcD and TAcD, respectively. The variation in VFA, pH, and ammonia nitrogen levels demonstrated that TPAcD combines the advantages of both MAcD and TAcD, with a higher hydrolysis rate in the early stage under thermophilic conditions (55 °C) and a suitable environment in the later stage under mesophilic conditions (35 °C). The kinetic parameters of anaerobic co-digestions also demonstrated that TPAcD performs better. Therefore, further research on TPAcD of sewage sludge and food waste is warranted due to its significant improvements in methane production rate, total methane yield, and system stability. Additionally, TPAcD contributes to reducing carbon emissions and supports the realization of “carbon neutrality”.

Hydrothermal carbonization of sugar beet pulp: optimization and characterization

AbstractIn this study, the optimum hydrothermal conditions of sugar beet pulp were investigated by Response Surface Methodology (RSM) based on Central Composite Design (CCD). The hydrochar obtained from sugar beet pulp (SBP) was optimized for maximum yield and carbon content. Process conditions were chosen with reaction temperatures of 200–240 °C, residence time 60–150 min, and biomass to water ratio of 1:3–1:10. The yield and carbon content of the hydrochar varied with the process parameters. In order to obtain hydrochar with the highest yield and carbon content in optimization, the reaction temperature should be 220.74 °C, the biomass to water ratio should be 1:3, and the residence time should be 95.58 min. High heating value, energy and mass yield, and energy densification ratio of sugar beet pulp and hydrochar were also investigated. The products were characterized using FT-IR, SEM, and ultimate analysis techniques. The Coats-Redfern method was used to estimate the kinetic parameters of the combustion processes. The activation energy values of SBP and SBP-HC products were calculated as 13.88 and 11.46 kJ/mol, respectively. The kinetic data were used to determine the thermodynamic parameters (ΔH, ΔG, and ΔS). As a result, the properties of hydrochar produced from sugar beet pulp under optimum conditions have been extensively investigated and the results have shown that hydrochar has potential for use in different areas.

The influence of drug-induced metabolic enzyme activity inhibition and CYP3A4 gene polymorphism on aumolertinib metabolism

The purpose of this study is to clarify the drug interaction profile of aumolertinib, and the influence of CYP3A4 genetic polymorphism on aumolertinib metabolic characteristics. Through microsomal enzyme reactions, we screened 153 drugs and identified 15 that significantly inhibited the metabolism of aumolertinib. Among them, telmisartan and carvedilol exhibited potent inhibitory activities in rat liver microsomes (RLM) and human liver microsomes (HLM). In vivo, the pharmacokinetic parameters of aumolertinib, including AUC and Cmax, were significantly altered when co-administered with carvedilol, with a notable decrease in the clearance rate CLz/F. Interestingly, the pharmacokinetic parameters of the metabolite HAS-719 exhibited a similar trend as aumolertinib when co-administered. Mechanistically, both telmisartan and carvedilol exhibited a mixed-type inhibition on the metabolism of aumolertinib. Additionally, we used a baculovirus-insect cell expression system to prepare 24 recombinant CYP3A4 microsomes and obtained enzymatic kinetic parameters using aumolertinib as a substrate. Enzyme kinetic studies obtained the kinetic parameters of various CYP3A4 variant-mediated metabolism of aumolertinib. Based on the relative clearance rates, CYP3A4.4, 5, 7, 8, 9, 12, 13, 14, 17, 18, 19, 23, 24, 33, and 34 showed significantly lower clearance rates compared to the wild-type. Among the different CYP3A4 variants, the inhibitory potency of telmisartan and carvedilol on the metabolism of aumolertinib also varied. The IC50 values of telmisartan and carvedilol in CYP3A4.1 were 6.68 ± 1.76 μM and 0.60 ± 0.25 μM, respectively, whereas in CYP3A4.12, the IC50 exceeded 100 μM. Finally, we utilized adeno-associated virus to achieve liver-specific high expression of CYP3A4*1 and CYP3A4*12. In the group with high expression of the less active CYP3A4*12, the magnitude of the drug-drug interaction was significantly attenuated. In conclusion, CYP3A4 genetic polymorphism not only influences the pharmacokinetic characteristics of aumolertinib, but also the inhibitory potency of telmisartan and carvedilol on it.

Kinetic ITC of DNA Aptamers Binding for Small Molecules and Implications for Binding Assays and Biosensors.

The determination of kon and koff values through kinetic analysis is crucial for understanding the intricacies of aptamer-target binding interactions. By employing kinetic ITC, we systematically analyzed a range of ITC data for various aptamers. Upon plotting their kon and koff values as a function of their Kd values, a notable trend emerged. Across a range of Kd values spanning from 28 nM to 864 µM, the kon value decreased from 2´105 M-1s-1 to 96 M-1s-1, whereas the koff value increased from 1.03´10-3 s-1 to 0.012 s-1. Thus, both kon and koff contributed to the change of Kd in the same direction, although the range of kon change was larger. Since experiments are often run at close to the Kd value, this concentration effect also played an important role in the observed binding kinetics. The effect of these kinetic parameters on two common sensing mechanisms, including aptamer beacons and strand-displacement assays, are discussed. This work has provided the kinetic values of small molecule binding aptamers and provided insights into aptamer-based biosensors.

Combustion Behavior of Cellulose Ester Fibrous Bundles from Used Cigarette Filters: Kinetic Analysis Study

This study is focused on the detailed examination of the combustion properties and kinetic analysis of a cellulose acetate fibrous bundle (CAFB), separated from used cigarette filters. It was shown that the faster rate of CAFB heating allows a large amount of heat to be supplied to a combustion system in the initial stages, where the increase in heating rate has a positive response to ignition behavior. The best combustion stability of CAFB is achieved at the lowest heating rate. Through the use of different kinetic methods, it was shown that combustion takes place through two series of consecutive reaction steps and one independent single-step reaction. By optimizing the kinetic parameters within the proposed reaction models, it was found that the steps related to the generation of levoglucosenone (LGO) (by catalytic dehydration of levoglucosan (LG)) and acrolein (by breakdown of glycerol during CAFB burning—which was carried out through glycerol adsorption on a TiO2 surface in a the developed dehydration mechanism) represent rate-controlling steps, which are strongly controlled by applied heating rate. Isothermal predictions have shown that CAFB manifests very good long-term stability at 60 °C (which corresponds to storage in a sea shipping container), while at 200 °C, it shows a sudden loss in thermal stability, which is related to the physical properties of the sample.

Morphological changes and color development during cookie baking-Kinetic, heat, and mass transfer considerations.

Color and shape are important quality attributes in baked goods, particularly cookies. Composition and processing conditions determine and influence color development and morphological changes in these baked goods. The objective of this study was to systematically evaluate the evolution of color and shape during baking to determine useful correlations that can be implemented during the assessment and modeling of the baking process. Cookies (AACC-I standard protocol 10-53.01) were baked at 185, 205, and 225°C. Moisture content, water activity, surface temperature, characteristic dimensions (radius and thickness), and color indexes (lightness, redness, blueness, and browning index [BI]) were monitored at different locations on the cookie surface and baking times. Relationships among the tested conditions were explored using correlation analysis. The cookies' dimensions and color indexes were strongly correlated with changes in moisture content over time, and those relationships were characterized using empirical models. The temperature dependence of the kinetic parameters of the changes in lightness and BI was also described and deemed independent of the location on the cookie surface. This study provides insights into the influence of heat and mass transfer on the physical and physicochemical changes of cookies during baking. The kinetic and secondary models developed in this study can serve as important components for establishing a comprehensive approach for coupling heat transfer, mass transfer, and reaction kinetics to estimate and optimize cookie-baking processes. PRACTICAL APPLICATION: The findings from this study provide valuable information for better understanding the morphological changes and color developments during the cookie-baking process. The quantitative data and models generated in this study will allow identifying baking conditions for better quality development.

Vitrification of Human Oocytes Before or After Rescue-IVM Does not Impair Maturation Kinetics but Induces Meiotic Spindle Alterations.

Cryopreservation of in vitro matured oocytes is still considered as an experimental alternative to mature oocyte vitrification after ovarian stimulation. Here, we investigated whether rescue-IVM should be performed before or after vitrification. For this, 101 immature oocytes (germinal vesicle stage) from women undergoing ICSI were used. Oocytes were divided into three groups: freshly in vitro matured oocytes (IVM), freshly in vitro matured oocytes subsequently vitrified (IVM + VIT) and vitrified/warmed GV oocytes then in vitro matured (VIT + IVM). Oocyte maturation rates and kinetics were assessed using time-lapse technology. Spindle dimensions and polarity, chromosome alignment and cytoplasmic F-actin filament length and density were determined using confocal microscopy and quantitative image analyses. No differences in IVM rates (fresh IVM: 63.16% and IVM post-VIT: 59.38%, p = 0.72) and timings (17.73h in fresh IVM, 17.33h in IVM post-VIT, p = 0.72) were observed whether IVM is performed freshly or after vitrification. Meiotic spindles were shorter in VIT + IVM (10.47µm vs 11.23µm in IVM and 11.40µm in IVM + VIT, p = 0.012 and p = 0.043) and wider in IVM + VIT (9.37µm vs 8.12µm in IVM and 8.16µm VIT + IVM, p = 0.027 and p = 0.026). The length-to-width ratio was lower in vitrified groups (IVM + VIT: 1.19 and VIT + IVM: 1.26) compared to IVM (1.38), p = 0.013 and p = 0.014. No differences in multipolar spindle and chromosome misalignment occurrence and cytoplasmic F-actin filament length and density were observed between groups. Our results suggest vitrification before or after rescue-IVM does not seem to impair maturation rates and kinetics parameters but induces meiotic spindle alterations.

Characterization of tritiated JNJ-GluN2B-5 (3-[3H] 1-(azetidin-1-yl)-2-(6-(4-fluoro-3-methyl-phenyl)pyrrolo[3,2-b]pyridin-1-yl)ethanone), a high affinity GluN2B radioligand with selectivity over sigma receptors.

Here, we describe the characterization of a radioligand selective for GluN2B-containing NMDA receptors, 3-[3H] 1-(azetidin-1-yl)-2-(6-(4-fluoro-3-methyl-phenyl)pyrrolo[3,2-b]pyridin-1-yl)ethanone ([3H]-JNJ- GluN2B-5). In rat cortical membranes, the compound bound to a single site, and the following kinetic parameters were measured; association rate constant Kon = 0.0066 ± 0.0006 min-1 nM-1, dissociation rate constant Koff = 0.0210 ± 0.0001 min-1 indicating calculated KD = Koff/Kon = 3.3 ± 0.4 nM, (mean ± SEM, n = 3). The equilibrium dissociation constant determined from saturation binding experiments in rat cortex was KD of 2.6 ± 0.3 nM (mean ± SEM, n = 3). In contrast to the widely used GluN2B radioligand [3H]-Ro 25-6981, whose affinity Ki for sigma 1 and sigma 2 receptors are 2 and 189 nM, respectively, [3H]-JNJ-GluN2B-5 exhibits no measurable affinity for sigma 1 and sigma 2 receptors (Ki > 10 μM for both) providing distinct selectivity advantages. Anatomical distribution of [3H]-JNJ-GluN2B-5 binding sites in rat, mouse, dog, monkey, and human brain tissue was studied using invitro autoradiography, which showed high specific binding in the hippocampus and cortex and negligible binding in the cerebellum. Enhanced selectivity for GluN2B-containing receptors translated to a good signal-to-noise ratio in both invitro radioligand binding and invitro autoradiography assays. In conclusion, [3H]-JNJ-GluN2B-5 is a high-affinity GluN2B radioligand with excellent signal-to-noise ratio and unprecedented selectivity.

Rapid Determination of Kinetic Constants for Slow-Binding Inhibitors and Inactivators of Human Histone Deacetylase 8

The kinetics and mechanism of drug binding to its target are critical to pharmacological efficacy. A high throughput (HTS) screen often results in hundreds of hits, of which usually only simple IC50 values are determined during reconfirmation. However, kinetic parameters such as residence time for reversible inhibitors and the kinact/KI ratio, which is the critical measure for evaluating covalent inactivators, are early predictive measures to assess the chances of success of the hits in the clinic. Using the promising cancer target human histone deacetylase 8 as an example, we present a robust method that calculates concentration-dependent apparent rate constants for the inhibition or inactivation of HDAC8 from dose–response curves recorded after different pre-incubation times. With these data, hit compounds can be classified according to their mechanism of action, and the relevant kinetic parameters can be calculated in a highly parallel fashion. HDAC8 inhibitors with known modes of action were correctly assigned to their mechanism, and the binding mechanisms of some hits from an internal HDAC8 screening campaign were newly determined. The oxonitriles SVE04 and SVE27 were classified as fast reversible HDAC8 inhibitors with moderate time-constant IC50 values of 4.2 and 2.6 µM, respectively. The hit compound TJ-19-24 and SAH03 behave like slow two-step inactivators or reversible inhibitors, with a very low reverse isomerization rate.