The calculation of the number of ice crystals for the model of frazil ice evolution is very important and affects the whole frazil events. In this paper, the general formula for the number of frazil ice crystals was established considering secondary nucleation, flocculation, gravity and turbulent entrainment, and ice crystals by melting. Meanwhile, two physical processes of secondary nucleation and flocculation were expressed by introducing critical impact velocity and the probability of flocculation from previous models. It has been found that the simulation results of frazil ice evolution are in good agreement with the experimental data and actual project. Then, Sobol method is carried out to judge parameters’ influence degree, which found the number of nuclei produced  Frazil ice is the origin of other forms of ice, and it can develop surface ice to form ice cover and accumulate into hanging dams, or attach to river beds and canal bottoms to promote the growth of anchor ice. Frazil ice evolution is a complex physical process, involving the interaction of hydrodynamics, mechanical forces, etc. Therefore, it’s of great significance to research the frazil ice to mitigate or eliminate the aforementioned problems.For cold regions of the north, frazil ice formation and evolution are seriously affected the operational safety of water conveyance projects and rivers in winter. Affected by the weather, different degrees of icing problems often occurs, which will change the hydraulic conditions and cause damage to water conveyance structures7. It is generally believed that frazil ice evolution requires three conditions: turbulence, degree of supercooling and initial seed ice crystals5. The first two are necessary conditions for effectively maintaining the process of heat transfer between water and the external environment, while the latter is a key factor in heterogeneous nucleation. Many studies had found that frazil ice will appear and grow around 0 °C in nature9. In theory, temperature in nucleation never gets close to the critical temperature of heterogeneous nucleation of any known material10. Therefore, it is assumed that the initiation of ice crystals originated from the settlement of ice crystals in the air. The settlement of ice crystals is related to snowfall, blowing snow, water drop or bubbles produced by water collision12. Therefore, secondary heterogeneous nucleation is regard as the main mechanism of nucleation. Once the seed ice crystals appear, it will grow and multiply, and the latent heat released by the growing ice crystals and the heat exchange with the external environment between maintain a dynamic balance. The multiplication of ice crystals is mainly caused by many physical processes, such as the shedding of small ice crystals when ice crystals collide each other or solid boundaries, flocculation between crystals or turbulent shear fragmentation.Frazil ice evolution is mainly divided into two parts: the number of ice crystals and the heat balance. The former includes multiple physical process, such as initial seeding, secondary nucleation, flocculation, breakup, gravity and turbulent entrainment14, which can cause thunderstorms, or the accumulation of dry snow on the surface of cars and roads15. For the behavior of ice particles, the commonly used theoretical methods include the Hamaker theory, JKR  model, DMT (Derjaguin Muller Toporov) collision model, etc. These models adopt different assumptions to explain the adhesive interaction of spherical particles due to Van der Waals forces. The JKR contact theory is based on Hertz’s theory, which believes that the adhesion exists in the contact area16. And it is applicable to the case where the relative contact radius is large and the adhesion in outside the contact area is relatively small19. Using JKR theory of adhesive elastic contact to study the interaction between adhesion and inelastic deformation in the impact of elastic solids20.The secondary nucleation and flocculation of ice crystals are produced by the collision of solid particles. In fact, the collision of ice particles is very important in many physical systems. Ice particles in the atmosphere transfer electrical charge through collisions21. The flocculation of ice particles is divided into low-speed adhesion and high-speed fusion22, and the velocity of rivers or channels is generally low, so the ice crystals’ flocculation belongs to the former. In addition, adhesion of ice crystals at low collision velocities has been experimentally demonstrated, and there appears to be a critical velocity beyond which ice crystals no longer stick together24. The calculation formula of the critical velocity can be derived from the collision model. Blum et al.25 and Doan et al.26 compared the experimental data with the theoretical value of the model. They found that the relative collision velocity of SiO2 grains below 1.2 m/s often leads to adhesion, and rough grains are more prone to adhesion, which is consistent with the prediction of the theoretical model.Through a series of research experiments, it is found that the states of ice particles are divided into the unchanged state and the changed state, and present four different degrees of fragmentation after the secondary nucleation27 established the model of frazil ice formation and evolution based on the continuity equation of ice number and heat balance equation. Then he wrote the report about comprehensive overview of frazil ice28, and the dynamic evolution of the crystal size distribution function. Mercier29 using Monte Carlo methods and probability density function to simulate frazil ice formation. Base on Daly’s model, Wang7 divided the ice particles radius into different intervals to establish heat balance equation and number continuity equation. In these models, the process of secondary nucleation and flocculation is simply described, and its collision theory is not elaborated in detail. Shen et al.12 presented a numerical model based on a two-layer ice transport formulation for surface and suspended ice, and verified the calculation result of the model by the field data. Lindenschmidt30 and Blackburn et al.31 improved the model RIVER to simulate the dynamic processes of frazil ice in river systems with complex flow patterns. Makkonen10 established the model for the entire active frazil event. Although the relationship between the sizes of ice particle and heat balance was established, the change process of the number of particles was not well simulated.Combined with the above research results, a number of mathematical models have been developed to simulate all kinds of physical processes and frazil ice evolution. Daly27 proposed a theoretical formula for the rate of secondary nucleation based on the kinetics of secondary nucleation. Evans et al.32 and Mercier29 proposed the model for the kinetics of secondary nucleation, and considered that the collision breeding was the primary mechanism. Then Jucha et al.33 found that the impact of turbulence velocity fluctuation becomes the main factor determining the collision rate. Hammar and Shen5 simulated secondary nucleation and flocculation based on binary collisions of frazil particles. It can be found from that the quantization of each process is relatively complex, and many uncertain parameters can hardly guarantee the accuracy of model results. In addition, there is a lack of sensitivity analysis of these parameters to determine their position and role in the model.The number of particles is difficult to be quantified, because the behavior of collisions between ice crystals is not fully understood. Daly34 to judge parameters’ influence degree and analyzes the sensitivity of parameters to expound the influence of the supercooling process, which provides technical supports for the verification of model parameters.In this paper, based on previous models and the theory of collision between ice particles, two processes of secondary nucleation and flocculation were expressed by introducing critical impact velocity and probability of flocculation, and general formula for calculating the number of frazil ice crystals was established. Meanwhile, the method were verified though the experimental data and actual project combination with the two-layer ice transport model. Finally, this paper uses Sobol methodAccording to the available research results, in order to express the parameters clearly and calculate the model conveniently, in this paper, the frazil ice crystal is regarded as a uniform size of disc-shaped. The mean diameter of frazil ice crystals is used as the calculated size of all ice crystals. Also, assumed that the space of model is fully homogeneous and there is no difference with location.27 general equation and Svensson and Omstedt’s35 model, particles breakup is generally not considered because it only exists in large, weakly bounded aggregates at high shear29.Based on Daly’sBefore the water is supercooled no crystals can exist in the water, in fact, new crystals are introduced by seeding, ice crystals that fall into the water from the atmosphere. Therefore, the change of the number of ice crystals in the period of supercooling only considers five physical processes, namely initial seeding, secondary nucleation, flocculation, gravity and turbulent entrainment, and ice crystals by melting. The general formula that can calculate the number of frazil ice crystals are considered as32 and Mercier29 proposed the model for the kinetics of secondary nucleation, and it assumed the collision frequency was related to turbulent shear and differential rising. The number of particles produced by the collision of two identical ice crystals isSecondary nucleation and flocculation are both caused by the collision of ice crystals. Secondary nucleation is responsible for producing small particles by removing the nuclei created from the surface of the parent particles. Evans et al.7; Due to the mentclass2pt{minim29, assuming that the proportion of collisional energy transfer to the crystals per unit volume is 24, it is found that after millimetre-sized ice particles collide with each other, coefficients of restitution were evenly spread from 0.08 to 0.65, and 0.08% to 17%, 4% to 41% of the kinetic energy was converted into the particles’ rotation and translation. Therefore, the value range of the parameter According to the model established by MercierGrowing frazil ice crystals can release latent heat to make the water temperature increase and the growth rate of ice decrease. An active frazil event involves a negative feedback mechanism. This puts a limit on the amount of ice that can form, given the time and the heat flux from the water body. So it is quite logical to limit 35, Clarks36, Wang7, etc.Before the water is supercooled no crystals can exist in the water, therefore 23. The flocculation between ice crystals is closely related to the thermal conditions determined by water temperature. It is necessary for the temperature of the water to be less than 0 °C for flocculation to occur.Flocculation is an adhesion phenomenon caused by the adhesion of contact surfaces37 suggested the probability of flocculation is linearly related to the diameter of colliding particles. Some studies show that the adhesion between particles is related to the collision velocity, but most of the critical adhesion velocity is calculated in the air fluid. Although the ice crystal collision exists in the liquid fluid, there may be some differences. However, JKR theory, whose research object is collision contact surface, can effectively avoid the influence of fluid force. According to the JKR theory  was was19) wFigure According to the thermal balance process of frazil ice growth, the equation isDifferentiating Eq.  gives21\The latent heat flux released by the growth of all particles is equal to the heat flux into water, so that.The Nusselt number is a dimensionless number related to Prandtl number  to  to \docu35 were used to verify the accuracy and reliability of the model. Selecting the laboratory experiment no. 63 and no. 86 by Ye et al. as the examples and their characteristics of the supercooling processes shown in the Table Various experimental studies on frazil ice formation have been carried out in different laboratory setting. By changing the hydraulic and environmental conditions that include water depth, and water velocity, air temperature, different working conditions are simulated, so as to explore the variation law of each factor in the whole supercooling process. In this paper, experimental data of Ye et al.MAE (mean absolute error) and RMSE (root mean square error), the calculation results of each parameter are shown in Table Then, using Eqs.  and 26)26) to caFigure MAE and RMSE of the three parameters in the two experiments are very small, indicating that the errors with the measure values are small and have good agreement with them. The model can better explain evolution of frazil ice concentration and water temperature. Through comparison, it is found that the MAE and RMSE of no. 86 is significantly lower than that of no. 63, it be due to inaccuracy in the empirical equations of the model or artificial errors in experiments. The Person correlation coefficient between water temperature and concentration are 0.62 and 0.16, diameter and concentration are 0.86 and 0.88. It suggests that water temperature and concentration is poorly correlated, and concentration and diameter is well correlated.Table 41 can not only simulate supercooling, suspended ice, surface ice discharges and other processes, but also consider the influence of ice crystal quantity in the conservation of thermal energy. In order to further verify the applicability of the general formula, based on the two-layer ice transport formulation model, this paper simulates the ice-water two-phase flow of channel. Water conveyance projects have been built in the cold areas in northwest China. In winter, the open-flow ice-water two-phase water transfer mode is adopted mostly to alleviate the problem of water shortage. Taking an open ice-water two-phase channel in Xinjiang as example, in winter 2003-01, observations on water temperature and the rate of freezing were made. Taking January 8(th) as a typical temperature process, other parameters do not change, the general formula for the number of frazil ice crystals only is introduced into Shen’s model to simulate the ice process at 3:00, and the calculation results are compared with those Shen’ model was used, as shown in Fig. According to the characteristics of various models and the actual situation of the projects, the two-layer ice transport model proposed by Shen et al.MAE and RMSE of water temperature and the rate of freezing for the two model are calculated, as shown in Table The water temperature and the rate of freezing along the lines are shown in Fig. MAE and RMSE of water temperature and the rate of freezing using this method is smaller, which proves the feasibility of the formula. The MAE and RMSE of introducing the general formula are smaller than those of Shen’ model, which it further shows that the general formula for the number of frazil ice crystals can improve the accuracy of simulation results to a certain extent.The numerical simulation results introduced the general formula are in agreement with the field observations in Fig. 48 Suppose the model can be represented as In the process of simulation, although the selection of some parameters refer to some relevant literature, it will still have a certain impact on the simulation results. In order to better understand the role of these parameters, special methods should be adopted for analysis. Sobol method is a global sensitivity analysis method based on variance decomposition. Its core is to decompose the total variance of the objective function into the variance of a single parameter and the variance generated by the interaction between parameters by Nandakumar et al.For parameters, the first-order, second-order and total sensitivity are respectively: According to above model, the maximum supercooling is selected as the objective function, and the three model parameters:Figure Sensitivity analysis is one of the common methods to analyze uncertainty. Hence, the sensitivity analysis of three parameters—In the above two experiments for no. 63 and no. 86, keeping other parameters unchanged, the different values of 29. Due to the large difference in Reynolds numbers between the two experiments for no. 63 and no. 86, so the magnitude of parameter values varies greatly.Figure In practice, the number of ice crystals Figure In addition, it can also be seen that the influence of The process of secondary nucleation and flocculation are important for frazil ice formation and evolution. The establishment of general formula for the number of ice crystals can make the model comprehensive and accurate by introducing new parameters.The simulation results agreed favorably with observational data and actual project, and correspond with the theoretical supercooling process. It confirmed that the simulation can reflect the entire physical process of frazil ice formation and evolution.Through the Sobol analysis of the three parameters (In the sensitivity analysis of the three parameters, it shows that the initial number of ice crystal In this paper, through defining the processes of secondary nucleation and flocculation by introducing critical impact velocity and the probability of flocculation, the general formula for the number of frazil ice crystals is established and applied the model for frazil ice formation and evolution. The example analysis and sensitivity analysis are carried out for the model. Based on the above, the following conclusions can be drawn:Finally, in the future work, we will strengthen the relevant experiments of physical model and enrich the collection of measured data to obtain the internal relationship among many parameters, So that the parameters can be better calibrated to properly evaluate model.


Six female pigs received an intravenous bolus infusion of 4 g/0.5 g piperacillin/tazobactam over 30 min every 6 h. Steady state was assumed achieved in the third dosing interval (12–18 h). Microdialysis catheters were placed in a cannulated screw in the proximal tibial cancellous bone, in cancellous bone next to the screw, and in cancellous bone on the contralateral tibia. Dialysates were collected from time 12 to 18 h and plasma samples were collected as reference. For the low piperacillin target (8 µg/mL), comparable mean fT>MIC across all the investigated compartments (mean range: 54–74%) was found. For the high target (16 µg/mL), fT>MIC was shorter inside the cannulated screw (mean: 16%) than in the cancellous bone next to the screw and plasma (mean range: 49–54%), and similar between the two cancellous bone compartments. To reach more aggressive piperacillin fT>MIC targets in relation to the implant, alternative dosing regimens such as continuous infusion may be considered.Implant-associated osteomyelitis is one of the most feared complications following orthopedic surgery. Although the risk is low, sufficient antibiotic protection of the implant surface is important. The aim of this study was to assess steady-state piperacillin concentrations in the proximity of an orthopedic implant. Time above the minimal inhibitory concentration ( Implants have become a cardinal component of modern orthopedic surgery . The assImplantation of a foreign material increases bacterial virulence, as fewer bacteria colony forming units are required to induce a manifest infection compared to tissues free of implants ,16. TherfT>MIC) at the target site [fT>MIC. As the introduction of implants in bone tissue causes microstructural bone damage affecting the local extracellular environment, it is important to assess the effect of these mechanisms on proximate implant piperacillin concentrations [Piperacillin is a broad-spectrum get site ,20. Pipeget site ,22,23. Gget site ,14, whertrations ,21,23. Ttrations .fT>MIC in the proximity of an orthopedic implant following intravenous bolus infusion in pigs. A cannulated screw was applied, giving the opportunity to evaluate concentrations from both the inner and outer layer of the implanted screw.In this study, we used microdialysis to assess piperacillin steady-state All six pigs completed the study. With the exception of one cancellous bone catheter next to the screw, data were obtained from all the catheters. The mean relative recovery (SD) was 27.5% (7.5) for the cannulated screw, 38.5% (15.9) for the cancellous bone next to the screw, and 59.8% (17.7) for the contralateral cancellous bone.fT>MIC values (minutes and percentages) for all compartments are shown in fT>MIC was shorter for the cannulated screw compared to the cancellous bone next to the screw and plasma.Mean 12–18 h and Cmax were significantly higher, and plasma Tmax, and T1/2 were significantly shorter than in the remaining compartments, except for T1/2 for contralateral cancellous bone. Additionally, the cannulated screw differed from the remaining compartments (lower Cmax and longer T1/2). T1/2 for the contralateral cancellous bone was significantly lower compared to cancellous bone next to the screw.The calculated pharmacokinetic parameters are presented in fT>MIC across all the investigated compartments (mean range: 54–74%), while mean fT>MIC for the high target (16 µg/mL) was shorter inside the cannulated screw (mean: 16%) than in the cancellous bone next to the screw and plasma (mean range: 49–54%), and comparable between the two cancellous bone compartments.We investigated piperacillin steady-state concentrations following bolus administration in the proximity of an orthopedic implant in the form of a cannulated screw, allowing the evaluation of concentrations from both the inner and outer layer of the screw. For the low piperacillin target (8 µg/mL), we found comparable mean max than all the remaining compartments and the lowest AUC12–18 h, but it presented comparable mean fT>MIC for the low target (8 µg/mL) and only shorter mean fT>MIC for the high target (16 µg/mL) compared to cancellous bone next to the screw and plasma. Pharmacokinetically, this is mainly explained by a fast penetration (short Tmax), mean Cmax above 8 μg/mL, and a longer elimination (T1/2) compared to plasma and the cancellous bone compartments. A prolonged elimination is favorable for time-dependent antibiotics such as piperacillin as it prolongs target exposure. However, in a mechanical deadspace, as in the hollow center of a cannulated screw, a complex situation may arise since the immune system cannot be expected to assist the antibiotic effect as in normally blood perfused tissue. A deadspace is presumably a poorly vascularized void with low oxygen tension and pH, which may favour bacterial proliferation and biofilm formation [The cannulated screw demonstrated significantly lower median Cormation . A recenormation . AlthougfT>MIC piperacillin between the cancellous bone next to the screw and the contralateral tibia, did unexpectedly indicate that fT>MIC piperacillin is not affected by the screw-induced stress in the cancellous bone. In contrast to this, a recent study with a comparable setup, investigating co-administered vancomycin and meropenem concentrations in relation to a cannulated screw, suggested that the presence of the screw lowered the concentrations next to the screw in comparison to the contralateral cancellous bone, especially for vancomycin [For evaluation of antibiotic concentration and protection of the outer layer of an implant, the current setup is expected to mimic the true impact of screw insertion on microstructural bone damage and alterations on local perfusion, extracellular pressure, osmotic stress and inflammation. Interestingly, the comparable mean ncomycin . The invncomycin ,28. As sIAO involves complex interactions between the bacteria, the implant (biofilm formation), the antibiotic, and the host immune system ,19. WhenfT>MIC target to protect an implant remains unknown. In clinical settings not involving implant surgery, different approaches have been suggested, ranging from 40 to 70% fT>MIC in non-critically ill patients, to 100% fT>MIC or even 100% fT>5xMIC in critically ill patients [fT>MIC value of 55% and 16% for the low and high MIC target, respectively, while the cancellous bone next to the screw (outer layer protection) presented with 74% and 54%, respectively. To reach more aggressive targets in relation to the implant, a different dosing strategy may be needed. In the present study, the cannulated screw showed a prolonged T1/2 which is favorable as it prolongs target exposure, and thereby mimics continuous infusion to some extent. For piperacillin, application of continuous infusion has been thoroughly investigated, both in terms of clinical outcome and target tissue fT>MIC, demonstrating superior effect of continuous infusion [It may require higher antibiotic concentrations than the employed planktonic targets to gain full antibiotic protection of the implant in orthopaedic surgery. The optimal piperacillin patients ,33. In tinfusion ,34. TherThe main limitation in the present study was the low number of pigs (n = 6), which may affect study power. However, the frequent microdialysis sampling fully utilizes the potential of the setup and increases the reliability of the reported data. For clinical relevance, piperacillin was administered in combination with tazobactam; however, tazobactam concentrations were not obtained. Previous studies have shown a comparable pharmacokinetic profile of piperacillin when administered alone and together with tazobactam ,36. TherThis study was performed at the Institute of Clinical Medicine, Aarhus University Hospital, Aarhus, Denmark. Chemical analyses were performed at the Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark. The study was conducted in accordance with ARRIVE and approved by the Danish Animal Experiments Inspectorate (License No. 2017/15–0201-01184) and carried out in accordance with existing laws and guidelines. To meet the 3 Rs by reducing the number of animal used, the same pigs have provided data to another study with a different purpose [Microdialysis is a catheter-based method with a precision pump connected to the inlet of the catheter which has a semipermeable membrane at the tip. At the outlet of the catheter, there is a collecting vial. Sampling is based on passive diffusion of unbound molecules across the membrane along the concentration gradient . As the Six female pigs  were included. The pigs were anesthetized by a combination of propofol  (550 mg/h) and fentanyl  (0.6 mg/h). Temperature (range 36.3–38.3 °C) was monitored by a rectal thermometer and regulated with blankets or icepacks. pH (range 7.38–7.53) was monitored through arterial puncture and regulated through ventilation. With the pig in a supine position, the proximal part of the left tibia was assessed through an anteromedial incision. By fluoroscopic guidance, a Kirshner wire (K-wire)  was drilled into the cancellous bone distal to the epiphysial plate. The K-wire placed in the left bone was used as guidance to drill a 35 mm hole performed by a cannulated drill. Afterwards, the cannulated drill and K-wire was removed in order to insert a 35 mm cannulated screw (Ø 6.5). Approximately 2–4 mm next to the cannulated screw, a cancellous drill hole was performed in a parallel direction in the axial plane . A similar drill dole was performed on cancellous bone in the contralateral leg . To avoig for 10 min. Afterwards, plasma samples were stored at −80 °C until analysis. Piperacillin was administered as a 4 g bolus infusion in combination with 0.5 g tazobactam (Fresenius Kabi) over 30 min every 6 h. Steady state was assumed achieved in the third dosing interval (12–18 h). A previous study observed a plasma half-life of 74 min (95% CI: 58–90) of piperacillin, suggesting steady state to occur within 5 to 7.5 h . After pUnbound piperacillin concentrations in the microdialysates and plasma samples, and benzylpenicillin concentrations in the microdialysates, were simultaneously quantified using ultra-high performance liquid chromatography with UV detection. For piperacillin, the total imprecisions (CV) were 4% at 8 μg/mL and 2% at 100 μg/mL in 0.9% NaCl solution, and 6% at 5 μg/mL and 9% at 80 μg/mL in plasma. For benzylpenicillin, CV were 5% at 2 μg/mL and 3% at 8 μg/mL in a 0.9% NaCl solution. The lower limit of quantification (LLOQ) was estimated at 0.1 μg/mL (CV% = 18%) for piperacillin and at 0.1 μg/mL (CV% = 11%) for benzylpenicillin. The calibration curve was only accepted if the correlation coefficient was >0.98. A more detailed description of the quantification method has previously been described ,24.fT>MIC using linear interpolation for each animal and compartment. Piperacillin clinical breakpoint for Pseudomonas aeruginosa (16 μg/mL = high target) and Enterobacterales (8 μg/mL = low target), published by the European Committee on Antimicrobial Susceptibility Testing (EUCAST), were used to evaluate fT>MIC [Microsoft Excel was used to estimate the e fT>MIC .12–18 h), peak drug concentration (Cmax), time to Cmax (Tmax), tissue penetration ratio (AUCtissue/AUCplasma), and half-life (T1/2). AUC12–18 h was calculated using the linear up-log down trapezoidal rule.Pharmacokinetic parameters were determined separately for each pig and compartment by non-compartmental analysis using STATA . Due to the small sample size, normal distribution was not achieved. Therefore, conversion into logarithmic transformation was necessary in order to reduce skewness of the original data. As a result, pharmacokinetic parameters are computed as medians. The following pharmacokinetic parameters were determined: Area under the concentration–time curves /λeq, where λeq is the terminal elimination rate constant estimated by linear regression of the log concentration on time. All variables were analyzed using a mixed model considering the variances between pigs. The model assumptions were tested by visual diagnosis of residuals, fitted values, and estimates of random effects. A correction for degrees of freedom due to small sample size was performed using the Kenward–Roger approximation method. Overall comparisons between the compartments were assessed using F-test followed by pairwise comparison using t-test. A p-value < 0.05 was considered statistically significant. No correction for multiple comparisons was applied.CfT>MIC was found across all the investigated compartments for the low piperacillin target (8 µg/mL), indicating equal theoretical protection of the inner and outer layer of the screw for this target. For the high target (16 µg/mL), the inner layer of the screw displayed shorter mean fT>MIC than in the cancellous bone next to the screw and plasma, and mean fT>MIC was comparable between the two cancellous bone compartments. To reach more aggressive piperacillin fT>MIC targets in relation to the implant, continuous infusion, or alternative dosing regimens may be considered.In summary, steady-state concentrations in relation to an orthopedic implant were evaluated for piperacillin therapy regimens. Comparable mean


H-benzimiazol-2-one (CHBI). The structure of CHBI was determined by X-ray diffraction (XRD). The inhibitory properties of CHBI were investigated in a 3.5 wt.% NaCl solution on pure copper using various electrochemical techniques such as potentiodynamic polarization curves (PDPs) and electrochemical impedance spectroscopy (EIS), as well as scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX), UV-visible spectroscopy, and theoretical calculations. The obtained results indicate that CHBI is an excellent inhibitor, exhibiting remarkable effectiveness with an inhibition rate of 86.49% at 10−3 M. To further confirm the extent of adsorption of the inhibitory molecule on the copper surface, density functional theory (DFT) and Monte Carlo (MC) simulation studies were conducted. The results of this study demonstrate the synthesis and characterization of CHBI as a corrosion inhibitor. The experimental and theoretical analyses provide valuable insights into the inhibitory performance of CHBI, indicating its strong adsorption on the copper surface.This study focuses on the synthesis, theoretical analysis, and application of the corrosion inhibitor known as benzimidazolone, specifically 1-(cyclohex-1-enyl)-1,3-dihydro-2 Organic inhibitors are an exceptionally effective and cost-efficient approach in the wide range of methods employed for corrosion prevention. This approach has gained widespread application for mitigating copper corrosion in multiple fields. Reviewing the existing literature unveils that the existence of heterocyclic compounds acts as active adsorption sites for facilitating the interaction between the corrosion inhibitor and copper through the exchange of electrons. In addition, heterocyclic compounds within aromatic structures exhibit effective corrosion inhibitory properties in a variety of corrosive environments ,2,3,4.Benzimidazolone is a heterocyclic aromatic organic compound consisting of the fusion of benzene and imidazolone rings. This heterocyclic framework serves as the foundational structure for a significant class of compounds, showcasing intriguing biological activities across a wide range of therapeutic domains. Recent research has found that benzimidazolone derivatives exhibit anti-HBV activities ,6 as welThe literature abounds with examples that underscore the versatility and effectiveness of benzimidazole and its derivatives as corrosion inhibitors. Their unique molecular structure and compatibility with various industries and environments have solidified their role as essential components in ongoing efforts to protect valuable assets and infrastructure from the ravages of corrosion ,17,18,19Benzimidazole and its derivatives have also been employed as corrosion inhibitors for mild steel in an environment containing 1 M HCl ,32. ThesIn the present study, the adsorption and inhibitive effects of CHBI on the corrosion of copper in a 3.5 wt.% NaCl solution were investigated. The corrosion inhibition effectiveness and adsorption characteristics of CHBI were examined through a combination of experimental methods, including Potentiodynamic Polarization analysis (PDP), Electrochemical Impedance Spectroscopy (EIS), and a morphological study using SEM-EDX. Additionally, a theoretical approach was employed, utilizing Density Functional Theory (DFT) calculations at the B3LYP/6–311G level of theory, along with molecular dynamics simulation methods. These comprehensive methods provided a deeper understanding of CHBI’s corrosion inhibiting capabilities and its interactions with the copper surface.1) and keto-ester (2) in refluxing xylene. The structure of CHBI was determined by analytical and spectroscopic analyses. The known CHBI was identified based on the spectroscopic data reported in the literature + m/z 215.1179, calcd for C13H14N2O, 215.1140. Elem. Anal. Calcd for C13H14N2O: C 72.87%, H 6.59%, N 13.07%. Found: C 71.63%, H 6.53%, N 13.54%.The CHBI was prepared according to a reported procedure . A mixtun of 5 h . The rea2 values using SAINT [The collection of single-crystal X-ray data was performed using a Bruker D8 VENTURE PHOTON 3 CPAD diffractometer with Cu-Kα radiation (λ = 1.54178 Å) at a temperature of 150 K. The intensity data obtained were converted to Fng SAINT . Subsequng SAINT . The strng SAINT . Afterwang SAINT . HydrogeIn this experiment, a highly pure copper electrode (with 99.9% purity) was immersed in a 3.5 wt.% NaCl solution, which served as a highly corrosive environment. To protect the copper from corrosion, CHBI was used as a corrosion inhibitor at various concentrations: 1, 0.1, 0.05, and 0.01 mM. Before conducting the tests, CHBI was introduced into a blank solution. Prior to the corrosion testing, the copper samples underwent a step-by-step polishing process using different grades of emery paper (ranging from 400 to 2500 grains). The samples were then rinsed with distilled water and anhydrous ethanol and left to dry at room temperature. The assessment of CHBI’s corrosion inhibition effectiveness was conducted by immersing the copper samples in the corrosive solution for a duration of 1 h. This allowed the researchers to evaluate the inhibitory performance of CHBI under the specified conditions.2 was utilized as the working electrode. Additionally, a saturated calomel electrode (SCE) was employed as the reference electrode, and a platinum electrode with a surface area of 1 cm2 was used as the counter electrode. A 3.5 wt.% NaCl solution was applied to the working electrode for 1 h at 298 K. Following the immersion, two electrochemical tests were conducted: EIS and PDP measurements. For the PDP measurement, the polarization curve was scanned from −1000 mV to +600 mV using a scan rate of 1 mV s−1. On the other hand, the EIS was performed across a frequency range spanning from 0.01 Hz to 100 kHz, with an amplitude of 10 mV [The electrochemical assessments were conducted using the potentiostat/galvanic PGZ 100 electrochemical workstation, employing the conventional three-electrode system . For thiof 10 mV ,70. The To understand the performance of CHBI from an electronic point of view, structural optimization was performed, and the electronic properties of CHBI were obtained using DFT implemented in Gaussian 09  with theThe frontier molecular orbitals (FOM), Highest Occupied Molecular Orbital (HOMO), and Lowest Unoccupied Molecular Orbital (LUMO) play a crucial role in the analysis of the chemical reactivity of organic molecules. They are also employed to investigate the inhibitory properties of molecules, as their adsorption is associated with the potential interaction between the HOMO and LUMO of the inhibitory molecule and the metal surface. The mode of interaction can be determined by a transfer of electrons, either by a donation of electrons from the molecule toward the metal, or by a retro-donation of electrons from the metal toward the inhibitor. For this purpose, based on the energies of the frontier orbitals, the reactivity of the inhibitor was studied from quantum chemical parameters such as the band gap (2)–(18)) ,76,77: force field.A molecular dynamics (MD) simulation was performed between the inhibitor molecule and the Cu(110) surface using a slab of 5 Å. Simulation boxes of dimensions 25.50 × 25.50 × 38.33 ÅIn this study, we synthesized the compound CHBI for copper corrosion inhibition. We found that CHBI is an excellent inhibitor of copper corrosion in a 3.5 wt.% NaCl solution. The inhibition efficiency increases with inhibitor concentration and reaches a maximum of 86.49% at These findings not only expand our understanding of CHBI’s role as a corrosion inhibitor but also highlight potential avenues for further research in corrosion protection and inhibitor optimization, holding promise for practical applications in protecting copper and related materials from corrosion.


The present study investigates the effect of prednisolone and its mechanism of action on the regulation of platelet function. The results demonstrated that prednisolone affects platelet function by inhibiting thromboxane A2 generation, this result suggested a role of prednisolone in platelet TxA2 generation. Consistently, prednisolone did not affect the 2-MeSADP-induced aggregation in aspirinated platelets, where the secondary wave of aggregation and secretion were blocked by eliminating the contribution of TxA2 generation by aspirin. In addition, thrombin-induced platelet aggregation and secretion were inhibited in the presence of prednisolone by inhibiting the positive-feedback effect of TxA2 generation on platelet function. Furthermore, prednisolone completely inhibited 2-MeSADP-induced TxA2 generation, confirming the role of prednisolone in TxA2 generation. Finally, Western blot analysis revealed that prednisolone significantly inhibited 2-MeSADP-induced cytosolic phospholipase A2 (cPLA2) and ERK phosphorylation in non-aspirinated platelets, while only cPLA2 phosphorylation, but not ERK phosphorylation, was significantly inhibited by prednisolone in aspirinated platelets. In conclusion, prednisolone affects platelet function by the inhibition of TxA2 generation through the regulation of cPLA2 phosphorylation, thereby shedding light on its clinical characterization and treatment efficacy in dogs with hypercortisolism in the future.Glucocorticoids have been commonly used in the treatment of inflammation and immune-mediated diseases in human beings and small animals such as cats and dogs. However, excessive use can lead to Cushing’s syndrome along with several thrombotic and cardiovascular diseases. Although it is well-known that glucocorticoids exert a significant effect on coagulation, the effect of cortisol on platelet function is much less clear. Thus, we aimed to study the effects of prednisolone, one of the commonly used glucocorticoids, on the regulation of platelet function using murine platelets. We first evaluated the concentration-dependent effect of prednisolone on 2-MeSADP-induced platelet function and found that the 2-MeSADP-induced secondary wave of aggregation and dense granule secretion were completely inhibited from 500 nM prednisolone. Since 2-MeSADP-induced secretion and the resultant secondary wave of aggregation are mediated by TxA Prednisolone is one of the most commonly used glucocorticoids in the treatment of various diseases including inflammatory and immune-mediated diseases. Prednisolone has the ability to reduce the recruitment of inflammatory cells including eosinophils, lymphocytes, mast cells, and dendritic cells by reducing the production of chemotactic mediators and adhesion molecules and the ability of inflammatory cells to survive . Due to Cushing’s syndrome is a disorder characterized by polyuria, polydipsia, muscle and skin atrophy, weight loss, truncal obesity, and hair loss . Excessi2 (TxA2) that enables the recruitment of circulating platelets, activates the recruited platelets, and forms the hemostatic plug in the presence of thrombin by converting fibrinogen to fibrin. Collagen and vWF induce platelet activation by the glycoprotein (GP) VI-mediated signaling pathway through phospholipase C (PLC) γ2 activation [2, and thrombin cause platelet aggregation by the activation of the PLCβ pathway. ADP stimulates Gq-coupled P2Y1 and Gi-coupled P2Y12 receptor-mediated signaling pathways [q and G12/13 through protease-activated receptors (PARs) [2 activates platelets through the thromboxane prostanoid (TP) receptor by coupling to Gq and G12/13 [2 generation is regulated by calcium binding to cytosolic phospholipase A2 (cPLA2) [2 generation is mediated by the P2Y12 receptor [2 then causes w positive-feedback effect on platelet functional response [It has been well-established that platelets play a crucial role in thrombosis and hemostasis and have been known to play a vital role in the development of blood-related disorders including diabetes and cardiovascular diseases in humans. Various signaling mechanisms are involved in the activation of platelets. Initially, platelets are activated by the exposure of collagen and vWF from the extracellular matrix upon vascular damage. Activated platelets further release adenosine diphosphate (ADP) and generate thromboxane Ativation . In contpathways . Likewiss (PARs) . TxA2 acd G12/13 ,19. In a (cPLA2) ,21. Throreceptor . The genresponse .Several studies have suggested the occurrence of hyper-coagulation in patients with Cushing’s syndrome whose blood cortisol levels are high ,24,25. T2 generation. We have further shown that 2-MeSADP- and thrombin-induced TxA2 generation and 2-MeSADP-induced cPLA2 phosphorylation are inhibited in the presence of prednisolone. In conclusion, prednisolone affects platelet function by inhibiting TxA2 generation through the regulation of cPLA2 phosphorylation.In this study, we demonstrated the effect of prednisolone on platelet function and its underlying mechanism using washed murine platelets. We have shown that prednisolone inhibits 2-MeSADP-induced platelet secretion and the resultant secondary wave of aggregation. In aspirinated platelets, prednisolone had no effect on the 2-MeSADP-induced platelet aggregation as well as secretion, suggesting that prednisolone exerts its antiplatelet effect by regulating TxA1 (PGE1), sodium citrate, prednisolone, and acetylsalicylic acid (ASA) were purchased from Sigma . Anti-phospho-cPLA2 (Ser505), anti-cPLA2, anti-phospho-ERK (Thr202/Tyr204), and anti-total-ERK antibodies were purchased from Cell Signaling Technology . HRP-linked secondary antibody was purchased from Santa Cruz Biotechnology . Thromboxane B2 (TxB2) ELISA kit was purchased from Enzo Life Sciences . All other reagents were of reagent grade.2-MeSADP, thrombin, apyrase, prostaglandin EThirty C57BL/6 mice were purchased from Orient Bio, Inc. at the age of eight weeks  and housed in a semi-pathogen-free facility. No more than 5 mice were housed per cage and mice were freely provided with food and water under constant conditions of temperature (23 °C) and humidity (60%). All animal procedures were approved by the Chungbuk National University Animal Ethics Committee (CBNUA-873-15-02).g for 10 min at room temperature (RT) and 1 µM PGE1 was added. For aspirin treatment, the PRP was treated with 1 mM ASA for 30 min at 37 °C. By centrifuging plasma at 400× g for 10 min, platelet pellets were obtained from the plasma and re-suspended in Tyrode’s buffer (pH 7.4) with 0.05 units/mL of apyrase. The platelet count was adjusted to 1 × 108 cells/mL.Blood was collected via cardiac puncture from healthy mice in 3.8% sodium citrate as described previously . PRP was8 cells/mL) at 37 °C in a Lumi-aggregometer  was measured to assess agonist-induced platelet aggregation while stirring at 900 rpm. Amounts of 250 nM, 500 nM, and 1000 nM prednisolone were pre-incubated for 5 min with washed platelets before stimulation with agonists. ATP release from platelets was assessed using a luciferin luciferase reagent to detect platelet dense granule secretion.The light transmission through a 250 μL sample of washed platelets (1 × 108 cells/mL) were stimulated for 3.5 min in a platelet aggregometer under 900 rpm stirring conditions at 37 °C and the reaction was halted by snap-freezing the sample in liquid nitrogen. Samples were stored at −80 °C until TxB2 analysis was performed. Briefly, samples were centrifuged at 3000× g for 10 min at 4 °C after being thawed at RT, and the supernatant was diluted (1:20) with buffer. The diluted samples were measured to evaluate the levels of TxB2 in duplicates using a TxB2 ELISA Kit , according to the manufacturer’s instructions.Washed platelets  at RT for 1 h, and membranes were incubated overnight with anti-phospho-cPLA2 (Ser505), anti-cPLA2, anti-phospho-ERK (Thr202/Tyr204), or anti-total-ERK antibodies diluted to a concentration of 1:1000 with gentle agitation. After 3 washes for 5 min each with Tris-buffered saline with 0.1% Tween 20, the membranes were incubated with appropriate secondary antibody at RT for 1 h. After washing, membranes were incubated with chemiluminescence substrate  for 5 min at RT, and immune reactivity was detected by using iBrightTM CL1500 .Washed platelets were activated with 2-MeSADP for 2 min at 37 °C under stirring conditions, and the reaction was stopped by adding 6.6 N perchloric acid. Platelet samples were washed with distilled water, re-suspended with sample buffer, and denatured for 10 min. Platelet samples were separated on a 10% SDS polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Nonspecific binding sites were blocked by SuperBlockp > 0.05).All statistical calculations were performed using Prism software (version 9.1). The data were presented as mean ± standard error (SE). Statistical significance was established using the one-way analysis of variance (ANOVA) followed by a post hoc Dunnett’s multiple comparison test. The normality test was conducted by using a Shapiro–Wilk test and data were found to be normally distributed (In order to determine the role of prednisolone in platelet function, we first stimulated the platelets with 2-MeSADP and measured the platelet aggregation and dense granule secretion. As shown in 2 generation [2 generation, we stimulated the platelets with 2-MeSADP in the presence of aspirin, which blocks the positive-feedback effect of the generated TxA2. As shown in 2 generation.Consistent with the previous result, prednisolone inhibited 2-MeSADP-induced platelet secretion and the resultant secondary wave of aggregation at both low and high concentrations of agonist A. It is neration ,33. In o2 when stimulated with a low concentration of thrombin [2 generation.To check the role of prednisolone in PAR-mediated platelet function, we stimulated the platelets with various concentrations of thrombin. As shown in thrombin ,35. Take2 generation, we stimulated the platelets with 2-MeSADP and thrombin and measured the amount of TxA2 release. As shown in 2 generation were significantly inhibited in the presence of prednisolone, confirming the inhibitory effect of prednisolone on TxA2 generation.To confirm the effect of prednisolone on TxA2 generation in platelets [2 generation by prednisolone, we stimulated the platelets with 2-MeSADP and measured the cPLA2 ERK phosphorylation in non-aspirinated and aspirinated platelets. As shown in 2 phosphorylation in both non-aspirinated and aspirinated platelets. However, 2-MeSADP-induced ERK phosphorylation was significantly inhibited by prednisolone only in non-aspirinated platelets, while prednisolone had no effect on 2-MeSADP-induced ERK phosphorylation in aspirinated platelets where there is no contribution of generated TxA2. These results confirm that prednisolone directly inhibits cPLA2 phosphorylation to regulate TxA2 generation in platelets.ERK phosphorylation is one of the most important upstream signaling molecules in TxAlatelets . To undeGlucocorticoids are one of the most commonly used steroid medications to treat numerous types of allergies, inflammatory diseases, autoimmune disorders, and cancers . Risks a2 [2 generation was blocked by aspirin treatment, prednisolone had no further inhibitory effect on 2-MeSADP-induced platelet aggregation and secretion compared to non-aspirinated platelets, suggesting that prednisolone inhibits the 2-MeSADP-induced secondary wave of aggregation and secretion through the inhibition of the TxA2 generation in platelets.Sex hormones, another type of steroid, have a well-established role in platelet function ,42,43. A2 . When th2 generation and the subsequent granule secretion play a role at low-thrombin concentration-induced platelet activation [2 generation. More importantly, we found that 2-MeSADP-and thrombin-induced TxA2 generation was significantly inhibited by prednisolone, confirming the effect of prednisolone on TxA2 generation.Thrombin is the most potent agonist that activates platelets via the PAR-mediated pathway . It has tivation . Therefo2 in several cells [2 is an upstream molecule of TxA2 generation that separates arachinonic acid, a precursor of TxA2, from phospholipids [2 and MAPKs has not been determined in platelets. In our study, we found that 2-MeSADP-induced platelet cPLA2 phosphorylation was completely inhibited by prednisolone in both non-aspirinated and aspirinated platelets. However, 2-MeSADP-induced ERK phosphorylation was significantly inhibited only in non-aspirinated platelets, whereas no additional inhibitory effect of prednisolone on ERK phosphorylation was observed in aspirinated platelets. It has been known that ERK plays a major role in TxA2 generation downstream of P2Y1, P2Y12, and PARs in platelets [2 can induce ERK phosphorylation by itself in platelets [2 generation by directly regulating cPLA2 phosphorylation, and that the effect of prednisolone on ERK phosphorylation is mediated by inhibition of TxA2 generation in platelets. High glucocorticoids have been shown to be effective in combination with first-line medication for some bleeding disorders because of their coagulation function [Glucocorticoids are known to inhibit cPLAal cells ,50. cPLAholipids . Glucocoholipids ,53,54. Hholipids . So far,latelets ,33,56. Flatelets . Thus, ofunction . It has function . Furtherfunction . In contfunction ,60. Our 2 generation through the regulation of cPLA2 phosphorylation. Moreover, it indirectly regulates generated TxA2-mediated ERK phosphorylation to control platelet functional responses. Overall, our study provides evidence that could be applied to the therapeutic use of prednisolone in coagulation and bleeding disorders as well as the clinical management of Cushing’s syndrome in dogs in the future.Cushing’s syndrome is a disease that severely affects both humans and dogs and is potentially life-threatening if it remains untreated . It sign


Finally, analysis of The Cancer Genome Atlas colon cancer dataset indicated that tumors with high SIX4 expression were significantly enriched in the Inflammatory Response pathway. SIX4 expression also correlated with expression of multiple IFN-stimulated genes, inflammatory cytokines, and CD8A. Taken together, our results implicate that SIX4 is a principal regulator of STING expression in colon cancer cells, providing an additional mechanism and genetic marker to predict effective immune checkpoint blockade therapy responses.The cGAS/STING cytosolic DNA-sensing pathway plays a significant role in antitumor immunity. Expression of STING is tightly regulated and commonly reduced or defective in many types of cancer. We have identified SIX4 as a significant regulator of STING expression in colon cancer cells. We showed that knockout of SIX4 decreased STING expression at the mRNA and protein levels while ectopic expression of SIX4 increased STING expression. Depletion of SIX4 led to attenuated STING activation and downstream signaling. Reexpression of SIX4 or ectopic expression of STING in SIX4 knockout cells reversed the effect. Ectopic expression of SIX4 enhanced DMXAA and cGAMP-induced STING activation and downstream signaling. Importantly, decrease of SIX4 expression substantially decreased tumor infiltration of CD8Our studies demonstrate that SIX4 is an important regulator of STING expression, providing a genetic marker or a therapeutic target to predict or enhance immune checkpoint blockade therapy responses in colon cancer. Cyclic GMP-AMP (cGAMP) synthase (cGAS) is a cytosolic DNA sensor that when activated generates cGAMP. cGAMP is a second messenger that triggers the stimulator of interferon genes (STING) adaptor to activate downstream signaling, including the Tank-binding kinase-1 (TBK1)/IRF3 and the IkB kinase of the NFκB signaling. Activation of IRF3 and NFκB induce the expression of type I IFNs and inflammatory cytokines and chemokines to initiate innate and adaptive immune responses . Moreovein vivo remains largely unknown.Because it is an important regulator of innate immunity, STING expression is tightly controlled. Previous work has demonstrated that STING is regulated at the transcriptional, posttranscriptional, and posttranslational levels . For exaSine oculis homeobox (SIX) 4 is a member of the SIX family of homeobox transcription factors . SIX4 exin vivo. The reduction of SIX4-dependent tumor clearance was associated with decreased tumor infiltration of CD8+ T cells. Analysis of The Cancer Genome Atlas (TCGA) colon cancer dataset is consistent with a role for SIX4 in controlling STING-dependent tumor clearance. Taken together, our studies demonstrate that SIX4 controls STING expression and activation in colon cancer cells, providing an additional mechanism and a potential clinical genetic marker to predict effective response of patients with colon cancer to immune checkpoint blockade therapies.Here we show that SIX4 is a key regulator of STING expression in colon cancer cells. Knockout of SIX4 significantly decreased STING expression at the mRNA and protein levels, whereas ectopic expression of SIX4 increased STING expression. Reducing SIX4 expression resulted in attenuated activation of STING signaling by STING agonists. Reexpression of SIX4 or ectopic expression of STING in SIX4 knockout cells reversed the effect. As expected, ectopic expression of SIX4 enhanced STING pathway activation. Importantly, depletion of SIX4 significantly reduced efficacy of tumor clearance mediated by an anti-PD-1 antibody in syngeneic immune competent mice Mycoplasma every 3 months with MycoAlert PLUS Mycoplasma Detection Kit .Mouse colon cancer cell lines, MC38 (RRID:CVCL_B288) and CT26 (RRID:CVCL_7256), and human colon carcinoma cell line, HT29 (RRID:CVCL_A8EZ), were purchased from ATCC. Human colon carcinoma cell line, TENN (RRID:CVCL_Y378), was established in Dr. Brattain's lab in 1981 . All cel2. MC38 and CT26 cells were cultured in DMEM supplemented with 10% FBS while HT29 and TENN cells in McCoy's 5A medium  with 10% FBS. Cells were passaged two to five times between thawing and use in the described experiments.Cells were maintained at 37°C in a humidified incubator with 5% COThe information of antibodies used in this study is as following: Anti-SIX4 , anti-STING , anti-phospho-STING , anti-TBK1 , anti-phospho-TBK1 , anti-ISG15 , anti-STAT1 , anti-phospho-STAT1 , anti-Actin . The STING activator DMAXX and cGAMP were purchased from InvivoGen .Whole-cell lysates were prepared in RIPA buffer  supplemented with a protease inhibitor cocktail . Equivalent amounts of protein were separated by SDS-PAGE and transferred to a Nitrocellulose membrane . Proteins were detected using an enhanced chemiluminescence system (LI-COR Biosciences).Total RNA was isolated, and reverse transcribed to cDNA. Q-PCR analysis was performed using PowerUp SYBR Green Master Mix . The primer sequences for mouse IFNβ, CXCL10, and STING are ATGAGTGGTGGTTGCAGGC-F, TGACCTTTCAAATGCAGTAGATTCA-R; AGTAACTGCCGAAGCAAGAA-F, GCACCTCCACATAGCTTACA-R and GGAACACCGGTCTAGGAAGC-F, TGGATCCTTTGCCACCCAAA-R, respectively. The primer sequences for human IFNβ, CXCL10 and STING are ATGACCAACAAGTGTCTCCTCC-F, GGAATCCAAGCAAGTTGTAGCTC-R; AGCAGAGGAACCTCCAGTCT-F, ATGCAGGTACAGCGTACAGT-R and CTTCACTTGGATGCTTGCC-F, CCCGTAGCAGGTTGTTGTAATG-R, respectively. Actin was used as an endogenous control.The Alt-R CRISPR-Cas9 System (Integrated DNA Technologies) was used, in which guide RNA (gRNA) contains crRNA with specific DNA target sequence and tracrRNA labeled with ATTO 550 (ATTO-TEC). Two crRNAs targeting SIX4 (ACAACTCCACTCGGAACTTC and CCTCGCACACGCAGGCGACA) were synthesized. gRNAs complexed with CAS9 protein were transfected into MC38 cells by electroporation. ATTO 550-positive cells were sorted into pools by flow cytometry the next day. One week later, cells were harvested and SIX4 expression was measured by Western blot analysis to determine knockout efficiency.Six4 cDNA is purchased from genomics online  and cloned into pCDH-CMV-MCS-EF1α-Puro Cloning and Expression Lentivector from System Biosciences . Human SIX4 cDNA was amplified from human cell genomic DNA by PCR and cloned into pCDH-CMV-MCS-EF1α-Puro vector. The expression vectors were cotransfeceted into 293T cells with psPAX2  and pMD2.G . Supernatant containing viruses was collected and infected into colon cancer cells.Mouse MC38 and CT26 cells were treated with 10 µg/mL of DMXAA for 6 hours. Cells were harvest for protein and RNA isolation. HT29 and TENN cells were transfected with 10 µg/mL of cGAMP using lipofectamine 3000 . Six hours later, cells were harvested for protein and RNA isolation.Cells were seeded in 6-well plates at 400 cells per well. After 2 weeks, colonies were stained with MTT and dissolved in DMSO. Relative cell numbers were determined by spectrophotometric analysis at 570 nm.6) were inoculated subcutaneously into the flank of 6-week-old C57BL/6J (RRID:IMSR_JAX:000664) mice on one side. Mice was randomly divided into two groups. On days 7, 10, and 13, each mouse was treated with 200 µg of either control IgG  or anti-PD-1 antibody  by intraperitoneal injection. Tumors were monitored and measured every other day. Tumor volumes (V) were calculated by the formula V = W2 × L × 0.5, where W represents the largest tumor diameter and L represents the next largest tumor diameter. Upon termination of the experiments, tumors were dissected out and photographed on the same scale.Mouse experiments involving animals were approved by the Ohio State University Institutional Animal Care and Use Committee (IACUC) and IACUC regulations were followed. Exponentially growing MC38 cells  × 100%].Formalin-fixed paraffin-embedded blocks of tumors were cut into 4-µm-thick tissue sections. After antigen retrieval using Antigen Unmasking Solution , tissue slides were blocked with 10% Goat Serum  diluted in 1X PBS, followed by incubation with an anti-CD8 antibody  overnight at 4°C. The slides were developed with ImmPACT DAB Substrate Kit  and counterstained with hematoxylin . Five fields/slide were randomly selected for quantification by ImageJ (RRID:SCR_003070). Percentage of CD8https://portal.gdc.cancer.gov/; ref. n = 471) were classified in SIX4high or SIX4low based on SIX4 expression using z-score. Gene matrix files were modified to comply with .gct file format and used as input file for gene set enrichment analysis  with 1,000 phenotype permutations using the MSigDb gene sets (RNA sequencing (RNA-seq) data for TCGA colon cancer dataset (TCGA-COAD) was accessed through the Genomic Data Commons Data Portal . Student t test.LI-COR Image Studio Software (RRID:SCR_015795) was used for Western blot image acquisition and quantification. At least two sets of Western blots for each experiment were analyzed and the ratios of intensity values of specified proteins and respective actin were calculated. Mean ± SD were plotted. Statistics analysis was performed in GraphPad Prism 5 (RRID:SCR_002798) using Student https://portal.gdc.cancer.gov/. All other data in this article can be obtained from the corresponding author upon reasonable request.The patient data analyzed in this study were obtained from https://jaspar.genereg.net/) to identify potential STING regulators. We ascertained SIX4 as a promising candidate that might regulate STING expression. CRISPR/Cas9 and two distinct gRNAs were used to knock out SIX4 expression in MC38 cells. We found that deletion of SIX4 significantly reduced STING expression at both mRNA and protein levels  would likely improve the clinical treatment efficacy.SIX4 expression has been shown to be upregulated in colorectal cancer . High SIOur studies showed that SIX4 knockout tumors grew similarly as the control tumors with control IgG treatment, indicating that SIX4 expression by itself has insignificant effect on tumor growth in syngeneic mouse model. However, anti-PD-1 treatment showed lower tumor clearance rate in SIX4 knockout group than in control group even though the treatment reduced tumor growth in both control and SIX4 knockout groups with tumors remaining. These observations suggest that the PD-1 antibody operates more efficiently in control tumors to eliminate cancer cells at the beginning of the treatment than in SIX4 knockout tumors and that knockout of SIX4 may not be able to completely deplete STING expression. Moreover, cancer cells that survived the anti-PD-1 treatment probably developed escaping mechanism(s) and continued to grow regardless of SIX4 expression. Identifying additional mechanisms by which cancer cells evade immune regulation is an important and active field of research. Ultimately, these studies may shed light on the mechanisms of patients’ resistance to immune checkpoint blockade therapies and facilitate development of strategies to overcome the resistance.In summary, we have identified SIX4 as a novel regulator of STING expression, which displays a tumor suppression function by increasing antitumor immunity. Knockout of SIX4 attenuated STING activator–mediated activation of STING/IFNβ signaling cascade and reduced efficacy of an anti-PD-1 antibody to eliminate tumor growth in immune competent mice. Data analysis of human patients with colon cancer support the hypothesis that SIX4 increases STING/type I IFN signaling and enhances inflammatory responses, which facilitates T-cell infiltration and antitumor immunity. SIX4 could regulate STING expression directly by binding to its promoter or indirectly through its target genes. Additional studies will be required to distinguish these possibilities. Nevertheless, SIX4 regulation of STING expression appears to play a significant role in colon cancer. Therefore, our studies add an additional layer to SIX4 function in regulating colon cancer progression and identify it as a potential target for enhancing immunotherapy.Supplemental Figure 1Supplemental Figure 1 shows the quantification of western blots shown in Figure 1A and 1B.Click here for additional data file.Supplemental Figure 2Supplemental Figure 2 shows the quantification of western blots shown in Figure 2A and 2B.Click here for additional data file.Supplemental Figure 3Supplemental Figure 3 shows the quantification of western blots shown in Figure 2C.Click here for additional data file.Supplemental Figure 4Supplemental Figure 4 shows the quantification of western blots shown in Figure 2D.Click here for additional data file.


We show that the proton pumping activity of AHA2 is stimulated by anionic phospholipids, especially by phosphatidylserine. This activation was independent of the cytoplasmic C-terminal regulatory domain of the pump. Molecular dynamics simulations revealed several preferential contact sites for anionic phospholipids in the transmembrane domain of AHA2. These contact sites are partially conserved in functionally different P-type ATPases from different organisms, suggesting a general regulation mechanism by the membrane lipid environment. Our findings highlight the fact that anionic lipids play an important role in the control of H+-ATPase activity.The activity of membrane proteins depends strongly on the surrounding lipid environment. Here, we characterize the lipid stimulation of the plant plasma membrane H Plasma membrane H+-ATPases belong to the P3 subfamily and are only found in plant, fungi, and protozoa . Theyl growth .+-ATPases found in the model plant A. thaliana, the isoform 2 (AHA2) is the most extensively studied. Phosphorylation of residues in the C-terminal R-domain of AHA2  can inhibit or induce binding of regulatory proteins that are believed to displace the autoinhibitory R-domains from the catalytic domain, resulting in activation of the pump . ,24. 4,24and 3W5C ,26,27,28at M8–10 ,30. Bothative MS . For theS. cerevisiae Pma1 with lipid raft domains and its interaction with sphingolipids, required for delivery to the plasma membrane . Partic ATPases ,34,35,36the pump ,37,38. W+-ATPases from maize, Zea mays, rice, Oryza sativa, and mung bean, Vigna radiata [+-pumps. Of the tested anionic lipids, PS showed the strongest stimulation of H+ pumping as compared to PA and PG, implying a specific lipid–protein interaction. Consequently, several regions with an increased anionic lipid density were identified using coarse-grain MD simulations of AHA2 embedded in different membrane compositions, each containing 10% of the anionic phospholipid PS in a background of neutral lipids. The identified sites were all located at the lipid–protein interface, and potential residues for anionic lipid–protein interaction sites were subsequently found by contact analysis. Of the five sites located at the cytoplasmic side, site B with M1K57 and M1K60 has previously been identified as possibly involved in the function and regulation of P-type ATPases [M1K60 also being conserved in the two yeast proton pumps S. cerevisiae Pma1 and N. crassa Pma1. The two residues have previously been identified in the report on the refined crystal structure of AHA2 by Focht et al. (2017) as possible interaction sites for anionic phospholipids. Furthermore, MD simulations showed this region to partition into the membrane interface, inducing a local depression speculated to facilitate solvent access to the H+ binding site (D684-N106 pair) [S. cerevisiae Pma1 [N. crassa Pma1, solved in the auto-inhibited E1 conformation [N. crassa Pma1, M1 is buried deeply within the membrane facilitating cytosolic access towards the H+ binding site, with K115, corresponding to M1K60 in AHA2, oriented to the lipid headgroup–aqueous interface as a so-called snorkeling residue [S. cerevisiae Pma1, where additionally the proton pumping cycle is found to involve movement of M1 towards the cytosolic leaflet of the bilayer in the E2P state [N. crassa Pma1 based on an SERCA homology model [Reconstitution of purified AHA2 into liposomes with defined lipid composition enabled the specific characterization of the potential role of lipids on the pump outside the complexity of the native environment. The proton pumping activity of the resulting AHA2-containing proteoliposomes was shown to vary depending on the presence of anionic lipids, with the strongest stimulation by PS. Within cells, PS is enriched along the secretory pathway, constituting 1.5 mol% of the glycerophospholipids in late secretory vesicles and 7 mol% in the plasma membrane, respectively . Additio radiata ,17,44, i ATPases . A segmet et al. 17 as posiae Pma1  and N. cormation . In N. c residue . A simil2P state . Such regy model . In thisA. thaliana Ca2+-ATPase ACA2, corresponding to M1K60 in AHA2, resulted in a deregulated pump with activity similar to the activated state [N. plumbaginifolia Pma2, corresponding to M1P68 in AHA2, which is conserved in P3 ATPases and is responsible for the 90° kink in the M1 speculated to facilitate solvent access to the H+-binding site [In addition to the structural data detailed above, mutational studies on residues located in the region of site B have also been reported. An E167K mutation in the related ed state . A similing site ,48. Thising site ,43. ConsM4D272A and M4D275A located adjacent to the lipid–protein contact residues at site C seems to be the only examples of mutagenesis studies of the putative anionic interaction sites identified in this work [M4D272 and M4D275 at the periphery of the contact site buried deeper into the membrane near a putative cation binding site identified by [+-binding site to the exoplasmic space. A similar role for the attraction of protons in the E1 conformation to the H+-binding site could also be envisioned for sites A2 and B.Along with the above-mentioned residues at site B, mutations of the his work . Howeverified by ,21,39. GS. cerevisiae Pma1 and N. crassa Pma1 revealed a hexamer arrangement, confirming previous reports on the oligomerization of P3 type ATPases [N. crassa Pma1 autoinhibition is enhanced or possibly even dependent on the hexamer arrangement [1, A2, and C could potentially be involved in mediating the formation of hexamers. In the structure of the S. cerevisiae Pma1, lipids in the exoplasmic leaflet were found to bind to M3/M7, at the interface between two monomer units, near the location of site C. Unfortunately, the lipid headgroups were not resolved, and therefore their exact composition remains unknown. MD simulations [N. crassa Pma1 identified two putative lipid binding sites near our site A1 (Site I) and site C (Site II) also located at the interface of two monomers. Whereas site I showed an accumulation of anionic PS, in accordance with the observations from the MD simulations on AHA2, site II showed preferred binding of PC. The identification of a putative PS binding site for N. crassa Pma1 fits well with reports showing the activity of the yeast proton pump to depend on the presence of anionic lipids [N. crassa Pma1 containing polymer nanodiscs [The recently reported structures of the yeast proton pumps  ATPases ,45,46,49 ATPases ,50. Even ATPases , while fc lipids  and the anodiscs . The effsn-glycero-3-phosphatidylcholine (PC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (PG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylethanolamine (PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (PA), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- (N-NBD-PE) were purchased from Avanti Polar Lipids Inc. . N-dodecyl-β-maltoside (DDM) and n-octyl-β-d-glucoside (OG) were obtained from Glycon . The ionophores valinomycin and CCCP, the pH-sensitive dye ACMA, and all other chemicals and regents were from Sigma-Aldrich , if not stated otherwise. ACMA was dissolved in dimethylsulfoxide; valinomycin in ethanol.Phospholipids 1-palmitoyl-2-oleoyl-A. thaliana H+-ATPase isoform 2 (designated AHA2), containing a hexahistidine (6 × His) and a SNAP® tag at the N-terminal end of the protein, was overexpressed in the Saccharomyces cerevisiae strain RS-72  and purified according to previously published protocols [−1 pepstatin. The cells were lysed by mixing with glass beads, and the protein was solubilized and purified with DDM at a detergent:protein (w/w) ratio of 1:3 using batch-binding to a Ni2+-NTA resin. The purified protein was finally concentrated to 1–10 mg mL−1 using centrifugal concentrators with a cut-off at 100 kDa , frozen in liquid nitrogen and stored at −80 °C in storage buffer containing 50 mM Mes-KOH (pH 7), 50 mM KCl, 20% (v/v) glycerol, 1 mM EDTA and 1 mM DTT supplemented with 0.04% (w/v) DDM until further use.A 73 amino acid C-terminal truncated version of rotocols . All bufv/v) and trace amounts (0.5 mol%) of fluorescent marker lipid N-NBD-PE in a glass tube. In addition to binary lipid mixtures, a ternary lipid mixture containing 45:45:10 PC:PE:PS was included in the set-up. The solvent was removed using a rotary evaporator . The lipid film (10 mg) was re-hydrated in 667 µL reconstitution buffer  by vortexing in the presence of a glass bead (5 mm diameter) for 5 min above phase transition temperature of the lipids , yielding a final lipid concentration of 15–20 mM. The vesicle suspension was further processed by five freeze-thawing cycles (90 s in liquid nitrogen followed by 90 s in a 60 °C water bath) and extrusion (21 times) through a stack of polycarbonate membranes (pore size 200 nm) using a mini-extruder (Avanti Polar Lipids). The vesicles made from PC:PE were unstable (lipids felt out of solution after short term storage), possibly because of an increased amount of PE in the membrane and the increased phase transition temperature. Thus, in this case, all steps (hydration and extrusion) were performed above 30 °C without storage at 4 °C. Liposome solubilization by OG was monitored by measuring light scattering of the liposome-containing solution at 600 nm using a fluorometer , thereby determining the ‘onset’ and ‘total’ solubilization conditions [Top is the starting plateau of solubilization curve , Bottom is the final plateau of solubilization curve , V50 is the point of inflection, and slope refers to the slope at the point of inflection.Liposomes were prepared by re-hydration of a thin lipid film, followed by freeze-thawing and manual extrusion. Briefly, binary lipid mixtures were prepared by mixing appropriate volumes of the lipid stock solutions in chloroform/methanol . The protein/lipid/detergent mixture was subjected to gel filtration  by centrifugation . The eluate was incubated for 60 min at room temperature with 50 mg of prewashed SM-2 Bio-Beads  under end-over-end rotation to ensure detergent removal.To facilitate the insertion of AHA2 into liposomes, preformed liposomes (4–5 mM) were solubilized by an amount of OG that was just sufficient to result in a half-maximal scattering change at 600 nm (‘Turning Point’). Purified AHA2 (12.5 μg) was added at a protein-to-lipid ratio of 1:60 . H+ pumping was initiated by the addition of MgSO4 , and the H+-gradient dissipated by the addition of 5 μM CCCP. Fluorescence quenching was recorded over a period of 600–1200 s at 480 nm  at 23 °C using a PTI-Quantamaster 800. Fluorescence traces were normalized to the intensity measured directly after addition of MgSO4. Recorded traces were analyzed with a customized python script with the slope of a linear curve fitted to the first 30 s after magnesium addition used as a measure for proton pumping activity. Traces that upon magnesium addition did not show a drop in fluorescence  were considered inactive and excluded from analysis.Huenching . Proteol® 488, New England BioLabs Inc., Ipswich, MA, USA) followed by 20 pmol membrane permeable SNAP dye  in reconstitution buffer supplemented with 1 mM DTT or with each dye separately. Samples were analyzed by SDS-PAGE using 12% gels and visualized on a ChemiDoc XRS Imaging System  using the Image Lab™ software (https://www.bio-rad.com/zh-cn/product/image-lab-software?ID=KRE6P5E8Z&WT_mc_id=211202033049&WT_srch=1&WT_knsh_id=cr180107&gclid=EAIaIQobChMI0eKynPLxgAMVlZhmAh2HUArSEAAYASABEgJPdvD_BwE) and pre-programmed option for Coomassie stained gels or Alexa488/Alexa647 fluorophores, respectively [AHA2 orientation in liposomes was determined by site-specific labeling of the SNAP-tag. Proteoliposomes  were incubated for 2 h either sequentially, first with 20 pmol membrane impermeable SNAP dye (10 µM SNAP-Surfaceectively .v/v/v). Detergent and lipid standards were chromatographed on the same plate and applied without prior extraction by chloroform/methanol. For visualization, plates were stained with primuline  and photographed under long-wave UV light (Biorad ChemiDoc XRS Imaging System).Phospholipid phosphorus was assayed after heat destruction in presence of perchloric acid by the method of Bartlett (1959) . To checp values < 0.05 were interpreted as statistically significant. Based on the results, significance was indicated as following; with no statistical significance (ns) or statistically significant; p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.To analyze the kinetic data, customized algorithms were developed using python version 3.8.8 . Data armartinize 2.6 and an elastic network was applied to stabilize the backbone conformation [2. The protein was embedded separately in two lipid bilayers with insane [2. Three 3 µs production runs were started from the equilibrated systems using different random seeds for generating the initial velocities of the particles at 310 K.For all computational studies the coarse-grain MARTINI 2.2 ,62,63,64ormation  by conneh insane , containh insane ,68,69 at−1, separately for the membrane plane (xy-plane) and its perpendicular axis (z-axis).The “New-RF” MD parameters recommended by de Jong et al.  were useOverall translation and rotation of the protein were removed prior to analyses by structural alignment to the starting structure of the simulations. Analyses were conducted for each trajectory separately, and for the concatenated trajectories of the three analogous replicas. Contacts between protein residues and lipid headgroups were analyzed with GROMACS 2020.1 and custom Python scripts. A contact was counted when at least two beads, one from the protein residue and one from a lipid headgroup, were within 0.55 nm from one another and no other lipid was closer to the protein residue. The contacts were normalized by the number of timeframes to obtain the probability of finding a protein–lipid contact at any time. Two-dimensional lipid headgroup densities were computed with the GROMACS tool gmx densmap (grid spacing: 0.02 nm), separately for each leaflet and lipid species. Lastly, three-dimensional lipid densities were computed with MD Analysis ,72 consi+-ATPase isoforms 1 and 2, respectively, from Arabidopsis thaliana. Q42932, for the H+-ATPase isoform 2 from Nicotiana plumbaginifolia. Q7XPY2, for the H+-ATPase from Oryza sativa subsp. Japonica. P05030, for the H+-ATPase isoform 1 from Saccharomyces cerevisiae. P07038, for the H+-ATPase from Neurospora crassa. P04191, for the SERCA pump from Oryctolagus cuniculus and Q4H132, for the Na,K-ATPase from Squalus acanthias.Protein sequences were downloaded from the UniProt knowledgebase database (access January 2021) and aligned with the tool Clustal Omega ,74. Acce


The inhibition of h15-LOX-2 by oleoyl-CoA was further determined to be allosteric inhibition with a iK of 82 +/− 70 nM, an α of 3.2 +/− 1, a β of 0.30 +/− 0.07, and a β/α = 0.09. Interestingly, linoleoyl-CoA (18:2) was a weak inhibitor against h5-LOX, h12-LOX, and h15-LOX-1 but a rapid substrate for h15-LOX-1, with comparable kinetic rates to free linoleic acid . Additionally, it was determined that methylated fatty acids were not substrates but rather weak inhibitors. These findings imply a greater role for acyl-CoAs in the regulation of LOX activity in the cell, either through inhibition of novel oxylipin species or as a novel source of oxylipin-CoAs.Lipid metabolism is a complex process crucial for energy production resulting in high levels of acyl-coenzyme A (acyl-CoA) molecules in the cell. Acyl-CoAs have also been implicated in inflammation, which could be possibly linked to lipoxygenase (LOX) biochemistry by the observation that an acyl-CoA was bound to human platelet 12-lipoxygenase via cryo-EM. Given that LOX isozymes play a pivotal role in inflammation, a more thorough investigation of the inhibitory effects of acyl-CoAs on lipoxygenase isozymes was judged to be warranted. Subsequently, it was determined that C18 acyl-CoA derivatives were the most potent against h12-LOX, human reticulocyte 15-LOX-1 (h15-LOX-1), and human endothelial 15-LOX-2 (h15-LOX-2), while C16 acyl-CoAs were more potent against human 5-LOX. Specifically, oleoyl-CoA (18:1) was most potent against h12-LOX (IC Fat and lipid metabolism is a complex process that involves the synthesis, breakdown, and transport of lipids throughout the body . Lipids 50 = 32 µM). The current work expands on this result, screening a larger family of acyl-CoAs against h5-LOX, h12-LOX, h15-LOX-1, and h15-LOX-2. This work provides new insights into the inhibitory mechanism of acyl-CoAs against LOX isozymes, potentially indicating an endogenous role for acyl-CoAs as LOX inhibitors, which could lead to the development of novel therapeutics targeting these enzymes.Lipoxygenases are a class of non-heme iron-containing dioxygenases that catalyze the oxygenation of polyunsaturated fatty acids, resulting in the formation of lipid mediators such as leukotrienes and lipoxins ,28,29. T50 values for palmitoyl-CoA (16:0), palmitoleoyl-CoA (16:1), stearoyl-CoA (18:0), linoleoyl-CoA (18:2), ɣ-linolenoyl-CoA (18:3), and docosahexaenoyl-CoA (22:6) indicated weak inhibition, with values greater than 200 µM , palmitoleoyl-CoA (16:1), linoleoyl-CoA (18:2), ɣ-linolenoyl-CoA (18:3), arachidonoyl-CoA (20:4), and docosahexaenoyl-CoA (22:6) indicated weak inhibition, with all values being greater than 50 µM , palmitoleoyl-CoA (16:1), linoleoyl-CoA (18:2), ɣ-linolenoyl-CoA (18:3), arachidonoyl-CoA (20:4), and docosahexaenoyl-CoA (22:6) indicated weak inhibition, with values greater than 100 μM , all inhibitor potency was lost.For h15-LOX-2, the ICn 100 μM . Stearoy50 values for ɣ-linolenoyl-CoA (18:3) and docosahexaenoyl-CoA (22:6) indicated weak inhibition, with values greater than 200 µM. Stearoyl-CoA (18:0), oleoyl-CoA (18:1), and linoleoyl-CoA (18:2) had slightly stronger inhibition, with IC50 values greater than 50 µM .In summary, the majority of the acyl-CoAs were not inhibitors against LOX isozymes; however, certain acyl-CoAs displayed micromolar potency. For h5-LOX, the C16 acyl-CoAs displayed the most promising inhibitory properties, with low micromolar potencies. However, with all the other isozymes—h15-LOX-1, h15-LOX-2, and h12-LOX—the C18 acyl-CoAs had the most potent inhibitory properties. Interestingly, adding double bonds to the acyl chain, such as from oleoyl-coA (18:1) to linoleoyl-CoA (18:2), eliminated significant inhibition potency, and complete loss of inhibitory activity was observed with γ-linolenoyl-CoA (18:3). This effect was most likely due to the added restriction on structural freedom with the additional double bonds, which would decrease flexibility and lead to lower binding affinity, thus reducing the availability of the proper orientation. With respect to biological relevancy, the most potent acyl-CoA interaction was that between oleoyl-CoA and h15-LOX-2, which displayed submicromolar potency. This level of potency drew us to undertake a more detailed kinetic analysis  and h15-LOX-1, as monitored by a change in absorbance at 234 nm . The kind values , suggest50 values greater than 200 µM for h12-LOX, h-15LOX-1, and h15-LOX-2. It should be noted that none of the methyl ester unsaturated fatty acids were substrates, except for methyl ester arachidonic acid and h15-LOX-1, as previously observed. However, the rate of this activity was low enough to still allow for IC50 measurements.The acyl-CoAs are large ester derivatives of fatty acids and, given this fact, it was deemed appropriate to determine if simpler esters could also inhibit the LOX isozymes. Thus, methyl-13Z,16Z-docosadienoic, methyl 13(Z),16(Z),19(Z)-docosatrienoate, and methyl docosatetraenoate were investigated and determined to be weak inhibitors, with IC50 potency against h15-LOX-2 (620 ± 60 nM), which led to further kinetic investigations at varying inhibitor concentrations. The replot of the apparent MK versus concentration for oleoyl-CoA exhibited a hyperbolic response with increasing amounts of acyl-CoA from 624 nM to 3.6 μM (Oleoyl-CoA displayed nanomolar ICo 3.6 μM . This hyMK (app) values were fitted with Equation 2 to yield a value for α of 3.2 +/− 1 and a value for Ki of 82 +/− 70 nM (catk/MK (app), which yielded a β value of 0.26 +/− 0.23 (i from the MK (app) plot to Equation 4 and fitted to the catk data (MK (α = 3.2 +/− 1) and catk (β = 0.30 +/− 0.07). This combined effect is emphasized in the β/α value, the allosteric effect on cat/KMk, which is significantly less than 1 (β/α = 0.09) and indicates cat/KMk allosteric inhibition. These allosteric kinetic data indicate the formation of a catalytically active ternary complex (I·E·S) between h15-LOX-2 and oleoyl-CoA and are consistent with our previous finding of an allosteric site for 13-(S)-HODE with 15-LOX-2 [i values from our hyperbolic fits (82 +/− 70 nM) were dramatically lower than those determined from IC50 measurements (620 ± 60 nM), which is consistent with the more accurate nature of the hyperbolic fits relative to the IC50 fits, due to the oversimplification of the IC50 analysis.Utilizing the equations derived from /− 70 nM . These t+/− 0.23 . Given tcat data . This fi15-LOX-2 . It shouInterestingly, in our recently published structure , oleoyl-Fatty acids used in this study were purchased from Nu Chek Prep, Inc. . All other solvents and chemicals were reagent-grade or better and were used as purchased without further purification.Reactions took place in an oxygen-deficient environment. DHA was added to an equimolar amount of butylated hydroxytoluene. Oxalyl chloride, along with 1 drop of dry dimethylformamide (DMF), was then added, and the mixture was stirred for 1 h at 37 °C and dried under vacuum. CoA was then added to a solution of tetrahydrofuran and 150 mM sodium bicarbonate in a ratio of 2.2/1.0. This solution was combined with the DHA solution for 30 min at 37 °C. The solution was dried to remove the tetrahydrofuran. Dichloromethane with 1.3% perchloric acid was used to extract the desired product away from the unreacted DHA acid. The water layer was retained and mixed with an equal volume of acetonitrile and lyophilized. The solid was washed with dry acetone and ether . The syng for 20 min at 4 °C. Saturated ammonium sulfate was added to achieve a final solution of 10% and inverted several times. The lysate was centrifuged at 40,000× g for 10 min at 4 °C. The pellet was discarded, and the supernatant was increased to a final concentration of 50% ammonium sulfate. The supernatant was rotated for 10 min at 4 °C then centrifuged at 40,000× g for 10 min at 4 °C. The pellet was split into 150 mg aliquots. The WT h5-LOX used for the kinetics in this work was not purified due to a dramatic loss in activity and was therefore prepared as an ammonium-sulfate precipitate.Overexpression and purification of his-tagged wild-type h12-LOX (Uniprot entry P18054) , h15-LOX50 values for each acyl-CoA were measured against h5-LOX, h12-LOX, h15-LOX-1, and h15-LOX-2. The reactions were carried out in 25 mM HEPES buffer (pH 8.00), 0.01% Triton X-100, and 10 μM AA for h12-LOX; 25 mM HEPES buffer (pH 7.5), 0.01% Triton X-100, and 10 μM AA for h15-LOX-1 and h15-LOX-2; and 50 mM HEPES buffer (pH 7.5), 100 µM EDTA, 50 mM NaCl, 200 µM ATP, and 10 μM AA for h5-LOX. IC50 reactions were implemented with h15-LOX-1 (0.125 μM), h12-LOX (0.300 μM), h15-LOX-2 (0.5 μM), and h5-LOX  at 22 °C in a 1 cm2 quartz cuvette containing 2 mL of the according buffer and 10 μM AA for no longer than 2 min to avoid any cleavage of the acyl-coenzyme A. Inhibitor concentrations ranging from 50 μM to 0.01 μM were used. All reactions were conducted using a Cary-UV Vis spectrophotometer . The fastest rates over 15 s intervals were recorded. IC50 values were obtained by determining the percent of inhibition of the enzymatic rate at ten inhibitor concentrations and plotting the rate against the inhibitor concentration, followed by a hyperbolic saturation curve fit. The saturation curve fits were performed in duplicate or triplicate, depending on the quality of the data. It should be noted that stability measurements of the acyl-CoAs indicated that there was no appreciable hydrolysis in the short timeframe of the kinetic measurements (approximately one minute). Through HPLC monitoring, we were able to determine a lack of degradation of the fatty acyl-CoAs over the first hour.IC50 values for h5-LOX was used for product determination. Turnover was monitored by absorbance at 234 nm.h15-LOX-2 (0.200 μM), h15-LOX-1 (0.125 μM), h5-LOX (0.300 μM), and h12-LOX (0.300 μM) were reacted with 30 μM of acyl-CoAs in 2 mL of 25 mM HEPES  at room temperature with ambient oxygen. The same buffer as used to determine IC2 quartz cuvette containing 2 mL of the according buffer for no longer than 2 min to avoid any cleavage of the acyl-coenzyme A. Using a Cary-UV Vis spectrophotometer, the fastest rates over 15 s intervals were recorded. Kinetic values were obtained by plotting the rate against the substrate concentration, followed by a hyperbolic saturation curve fit. The saturation curve fits were performed in triplicate and on separate days to ensure there was no bias. The equations used to find kinetic values were from previous studies [The reactions were carried out in 25 mM HEPES buffer (pH 7.5), 0.01% Triton X-100. Varying amounts of substrates from AA ranging from 50 μM to 0.01 μM and 0, 150, 300, 450, and 600 nM of 18:2 acyl-coA were used. Reactions were performed at 22 °C in a 1 cm studies .In conclusion, our previously reported cryo-EM structure for h12-LOX indicated an acyl-CoA bound to its active site, most likely oleoyl-CoA . Based o