Pedicle screw placement in patients with osteoporosis is a serious clinical challenge. The bone mineral density (BMD) of the screw trajectory has been positively correlated with the screw pull-out force, while the computer tomography (CT) value has been linearly correlated with the BMD. The purpose of this study was to establish an in vitro osteoporosis model and verify the accuracy and effectiveness of automated pedicle screw planning software based on CT values in this model.Ten vertebrae (L1-L5) of normal adult pigs were randomly divided into decalcification and control groups. In the decalcification group, the vertebral bodies were decalcified with Ethylenediaminetetraacetic acid (EDTA) to construct an in vitro osteoporosis model. In the decalcification group, automatic planning (AP) and conventional manual planning (MP) were used to plan the pedicle screw trajectory on the left and right sides of the pedicle, respectively, and MP was used on both sides of the control group. CT values of trajectories obtained by the two methods were measured and compared. Then, 3D-printed guide plates were designed to assist pedicle screw placement. Finally, the pull-out force of the trajectory obtained by the two methods was measured.P < 0.05). In the decalcification group, the MP trajectory CT value was 2167.28 ± 65.62 Hu, the AP trajectory CT value was 2723.96 ± 165.83 Hu, and the MP trajectory CT value in the control group was 2242.94 ± 25.80 Hu (P < 0.05). In the decalcified vertebrae, the screw pull-out force of the MP group was 48.6% lower than that of the control group (P < 0.05). The pull-out force of the AP trajectory was 44.7% higher than that of the MP trajectory (P < 0.05) and reached 97.4% of the MP trajectory in the control group (P > 0.05).After decalcification, the BMD of the vertebra decreased from − 0.03 ± 1.03 to − 3.03 ± 0.29 (Automatic planning of the pedicle screw trajectory based on the CT value can obtain a higher screw pull-out force, which is a valuable new method of pedicle screw placement in osteoporotic vertebre.The online version contains supplementary material available at 10.1186/s12891-022-05101-6. Osteoporosis is a metabolic bone disease characterized by low bone mass. It is a common disease in elderly people, especially in postmenopausal women, and can cause changes in the biomechanical properties of the vertebrae , 2. In cThe placement position of the pedicle screw is a crucial factor affecting the pull-out force of the screw, and its accuracy is related not only to malposition but also to the biomechanical function of the pedicle screw . PreviouThe application of navigation-guidance technology based on CT images can provide a holistic anatomical image of the surgical area through a single scan. It could prevent repeated fluoroscopy during the operation, and locate and track the relative position of surgical instruments and anatomical structures in real-time, thus avoiding pedicle perforation and effectively reducing the occurrence of nerve and vascular complications caused by pedicles . HoweverTo address these problems, this study used an automatic planning software to analyze CT data on an osteoporosis vertebra model to search trajectories of the highest CT value. Trajectories with non-perforating pedicles and non-piercing vertebrae were found to obtain a better effect of screw fixation. The in vitro osteoporosis model was used to verify its accuracy and effectiveness. Surgeons who find pedicle screw placement difficult in patients with osteoporosis may be interested in this research, which may provide them with a potentially effective solution to avoid more implants.Ten lumbar vertebrae were taken from mature pigs . All pigs were healthy, and none had been exposed to any factors that might affect BMD. All vertebrae were stripped of surrounding muscle tissue, ligaments, and periosteum. Parts of the spinous and transverse processes were excised and embalmed with 10% paraformaldehyde for 48 h. Micro-CT (μCT) was used for detection. Five of the 10 vertebrae were used as a decalcification group, and the other 5 were used as a control group. Five vertebrae were randomly selected from all specimens using Ethylenediaminetetraacetic acid (EDTA) decalcified fluid for decalcification. A 3 mm *10 mm hole was drilled in the upper and lower endplates of the vertebral body. During the decalcification process, a stepper motor was connected to these holes through a silicone tube, and EDTA was pumped at a rate of 1 ml/min. This procedure allowed the cancellous bone and cortical bone of the vertebrae to be decalcified simultaneously. Decalcification was performed for 2–3 weeks. The EDTA solution was replaced weekly, and the vertebrae were scanned weekly with μCT until the osteoporosis criteria were met.The BMD level was expressed by T value, T value = /standard deviation of normal BMD. Due to the linear relationship between the CT value and BMD, the CT value of normal vertebrae could be used as a substitute value for normal BMD. The formula was rewritten as T’ value = /standard deviation of normal vertebral CT value. Osteoporosis was defined when the T’ value ≤ − 2.5.Software automatic planning and manual planning were used for pedicle screw trajectory planning, respectively. Software automatic planning was used for the left pedicle, and manual planning was used for the right pedicle. All trajectories were required not to pierce the pedicle and anterior vertebral margin. To ensure the uniformity of biomechanical testing, the specifications of all planned trajectories were unified as 6 mm * 30 mm.Finav  was used for automatic planning based on CT values. The surgeon only needs to manually adjust the area of interest to mark the target vertebral body and the narrowest part of the pedicle and specify the screw diameter and length according to the recommended screw diameter and length calculated by the software. Then, the software can automatically calculate and plan the screw trajectory with the largest CT value . The sofThe artificially planned trajectory passed through the midpoint of the minimum section of the pedicle. The direction was parallel to the endplate on the vertebral body with an angle position the screw on the central axis of the pedicle. The same senior spine surgeon manually planned all trajectories.The position of the screw trajectory was measured on axial and sagittal CT views . Draw guMimics software  was used to analyze the μCT data of the osteoporosis model, the trajectory of the automatic planning and manual planning were analyzed, and the CT values of the trajectory obtained by different planning methods were measured.In this study, 3D printed guide plates were used to realize planned trajectories. Mimics and 3-Matic  software were used to import CT data, and 3D image reconstruction was conducted. The 3D printing guide plates were designed according to the screw entry points and stop point coordinates obtained by automatic and manual planning. The automatic planning trajectories were on the left side, and manual planning trajectories were on the right side. The designed 3D printing guide plates were imported into the 3D printer software, the printing parameters were adjusted, and the plates were printed.Pedicle screw placement was assisted by 3D-printed guide plates. A 2.5 mm Kirschner wire was used to turn the hole through the guide head of the 3D-printed guide plate to obtain the screw trajectory, and a pedicle screw was inserted along the trajectory . The same screw specifications were used to obtain comparable results in subsequent pull-out tests.After placement, X-rays, and CT were used to verify the position of the pedicle screw to ensure that the pedicle screw was completely located in the pedicle without penetrating the pedicle and the vertebral cortex. The screw placement error was measured by preoperative and postoperative CT images. Similar to 2.3, d1 and θ1 represent the actual screw position as measured by postoperative CT. The displacement error is (d1-d); the angle error is (θ1-θ).Each vertebra was fixed so that the long axis of the pedicle screw was coaxial with the direction of tension . The axial pull-out test was performed at a speed of 5 mm/min, and the force applied to the screw was recorded in displacement increments of 0.05 mm until the screw was completely removed. The maximum load encountered during the test was defined as the peak load (pull-out force) at the time of failure. The test sequence of the left and right pedicles was alternated with each specimen to eliminate the effect of the test sequence.P < 0.05 indicated a significant difference.SPSS  was used for statistical analysis, and the data were expressed as the mean ± standard deviation. The normality of the two sets of continuous variables was assessed by the Shapiro-Wilk test (W test), and the F-test was used to determine whether the overall variance of the two sets of continuous variables was homogeneous. If the data were normally distributed and the variance was homogeneous, A paired sample T-test was used for pairwise comparisons of decalcification group data, and an independent sample T-test was used for intergroup comparisons. P < 0.05), and the T’ value decreased from − 0.03 ± 1.03 to − 3.03 ± 0.29 (P < 0.05). All the vertebral bodies in the decalcification group met the standard of osteoporosis (T’ < -2.5) , while the axial distance was less than that in manual planning (p < 0.05)  Table . The autP < 0.05). It should be noted that the CT value of the manually planned trajectory in the control group was 2242.94 ± 25.8 Hu, which was higher than that in the decalcification group (P < 0.05) but lower than that in the automatically planned trajectory in the decalcification group (P < 0.05)   Table .Fig. 23DP < 0.05). The pull-out strength of the automatically planned trajectory was 44.7% higher than that of the manually planned trajectory (P < 0.05) and reached 97.4% of the manually planned trajectory in the control group. There was no significant difference between the two groups (P > 0.05)  Table .Pedicle screw fixation is a serious challenge in spinal surgery when confronted with osteoporosis. The in vitro osteoporosis experiment is the most direct and effective method to study the biomechanical properties of osteoporotic vertebrae. In this paper, the research was conducted by constructing in vitro models of osteoporotic pig vertebrae. In these models, the CT value and screw pull-out force of automatic trajectory planning based on the CT value and manual trajectory planning were evaluated and compared. The results showed that automatic trajectory planning was significantly better than manual trajectory planning in terms of screw pull-out force, which may suggest that automatic pedicle screw trajectory planning based on CT values is a valuable method for pedicle screw placement in osteoporosis.The best models for studying osteoporosis in humans are from human cadavers, which are difficult to study because of their limited availability. One possible approach is to study vertebral specimens from large animals –25. In tPedicle screw placement in patients with osteoporosis has always been a difficult problem in spine surgery. Existing solutions include cement augmentation screw placement, cortical bone track screw placement (CBT), placement of varied materials in the screw path to increase local tissue density, increasing the screw diameter, and changing the screw thread structure. However, these methods all have their limitations. Bone cement leakage is a common complication of bone cement augmented screws, with a leakage rate of 11.6–82.4%, which can cause serious complications such as nerve injury, vascular injury, pulmonary cement embolism, cardiac embolism and anaphylactic shock . In CBT,The development and validation of automatic pedicle screw planning systems based on preoperative or intraoperative CT are in line with the current development trend of digital and intelligent spinal surgery. Computer-assisted preoperative planning pedicle screw placement is a relatively fast process that can be performed without the automatic participation of a spine surgeon . The optP < 0.05), but both were within the allowable range [P < 0.01). It should be noted that the CT values of screw tracks automatically planned in the decalcification group were 21.4% higher than those manually planned in the control group (P < 0.01). The use of 3D printed guide plates to place pedicle screws was exactly accurate. The displacement and angle errors of the actual screw and planned screw positions showed no significant difference (P > 0.05). The screws were completely located in the cortical bone, there was no perforation of the pedicle or anterior vertebral margin, and no local microfracture occurred. In vitro tests, 3D-printed guide plates are a viable alternative in scenarios where computer navigation is not available. However, in clinical application, it is impossible for surgeons to completely remove the soft tissue on the surface of the vertebrae, which makes it difficult to accurately fix the 3D-printed guide plates in the corresponding positions, resulting in the actual positions of the inserted pedicle screws significantly deviated from the planned trajectories. Therefore, the combination of automatic planning and surgical navigation systems may be the best way to use the automatic planning system in clinical practice.In this study, the manual planning group adopted parallel placement technology, which inserted screw parallel to the upper endplate of the vertebral body. The sagittal angle was 3.23 ± 1.64°, while the automatic planning was 19.19 ± 1.87°, which was significantly different (le range . The autP < 0.05). It should be noted that although the CT value of the automatically planned trajectory in the decalcification group was 21.4% higher than that of the manually planned trajectory in the control group, the screw pull-out force was only 97.4% of that in the control group. This result may suggest that the BMD of the bone tissue around the screw trajectory also influences the pull-out force of the screw, which requires further finite element analysis to explain the reason. Despite this, the automatic plan increased the pull force by 44.7% compared with the manual plan in the decalcification group, which was close to the proportion of improvement in bone cement-reinforced screws (47%) [Biomechanical research also proves that the automatic planning method is effective. The screw trajectory planned according to the CT value obtained a larger screw pull-out force than the traditional manually planned trajectory (ws (47%) . These rThe software can export trajectory coordinates and match them with reconstructed CT images, which provides a basis for future association and matching with surgical navigation systems. In the future, combined with navigation systems or surgical robot technology, optimal trajectory planning and screw placement of the osteoporotic vertebral body can be achieved increasingly quickly, providing a new option for pedicle screw placement of the osteoporotic vertebral body.In this study, an in vitro demineralized osteoporosis model of a porcine lumbar spine was used, and the evaluation index was only the CT value, without considering microscopic parameters such as bone trabecular structure. The structure of the pig lumbar vertebra is different from that of the human vertebra and EDTA decalcification simulates not true osteoporosis but more osteomalacia. The applicability of the pedicle screw should be verified in the patient’s CT and cadaver osteoporosis models. Specimens treated with preservatives inescapability caused changes in the material properties of specimens. Therefore, using fresh specimens to further improve the accuracy of the experimental study is necessary. In the pedicle screw pull-out experiment, we simulated direct violent pull-out, which had limited agreement with the actual pull-out situation in the human body. The pull-out force could be measured again after the cyclic fatigue test. This study specifically to age groups, requiring follow-up studies on normal vertebrae. The sample size of the test was small, and subsequent studies with larger sample sizes are needed to confirm its accuracy and effectiveness. Different designs and populations demographics to further investigate the effect of this automatic planning software are also needed.In the osteoporosis model, automatic planning of the pedicle screw trajectory based on CT value software is safe and effective without causing pedicle screw puncture. Automatic trajectory planning can obtain a higher screw pull-out force, which is 44.7% higher than manual planning and even reaches a size similar to that of normal manual planning. This method may be a valuable new method of pedicle screw placement in osteoporotic vertebral bodies.Additional file 1.


IC50 = 2.5 ± 0.6 µM, iK = 2.4 ± 1.0 µM, inactk = 0.07 ± 0.01 min−1, inactk/iK = 0.03 min−1 µM−1. Chrysin caused the highest reduction of heme . None of the tested flavonoids showed pseudo-irreversible inhibition. Although the inactivation of the CYP3A4 enzyme is caused by interaction with heme, inhibitor-heme adducts could not be trapped. These results indicate that flavonoids have the potential to inhibit the CYP3A4 enzyme and interact with other drugs and medications. However, possible food–drug interactions have to be assessed clinically.Acacetin, apigenin, chrysin, and pinocembrin are flavonoid aglycones found in foods such as parsley, honey, celery, and chamomile tea. Flavonoids can act as substrates and inhibitors of the CYP3A4 enzyme, a heme containing enzyme responsible for the metabolism of one third of drugs on the market. The aim of this study was to investigate the inhibitory effect of selected flavonoids on the CYP3A4 enzyme, the kinetics of inhibition, the possible covalent binding of the inhibitor to the enzyme, and whether flavonoids can act as pseudo-irreversible inhibitors. For the determination of inhibition kinetics, nifedipine oxidation was used as a marker reaction. A hemochromopyridine test was used to assess the possible covalent binding to the heme, and incubation with dialysis was used in order to assess the reversibility of the inhibition. All the tested flavonoids inhibited the CYP3A4 enzyme activity. Chrysin was the most potent inhibitor:  Flavonoids are secondary plant metabolites that are consumed through vegetables, fruits, teas, wines, propolis, medicinal plants, and so on. These compounds aid organoleptic characteristics of foods  and are of interest due to biological properties affecting human health ,2. As noosylated . Based oosylated . The conosylated ,6.O-methylated flavone believed to be connected with the prevention of heart diseases [Acacetin is an diseases , presentdiseases , anti-pldiseases , and antdiseases ,11 effecdiseases . Other fdiseases . Apigenidiseases , anti-hydiseases , and antdiseases  to anti-diseases . Chrysindiseases . Chrysindiseases , showingdiseases  and antidiseases  effects,diseases . Pinocemdiseases ,19,20.Overall, the consumption of flavonoids and foods rich in flavonoids is linked with beneficial effects on human health . HoweverThe CYP3A4 enzyme is responsible for the metabolism of 33% of drugs . BesidesThe aim of this study was to investigate the inhibition kinetic of acacetin, apigenin, chrysin, and pinocembrin using nifedipine as a marker substrate of CYP3A4 enzyme activity. Furthermore, the aim of this study was to investigate the possible covalent binding of flavonoids to the heme part of CYP3A4, as well as to test their possible role in pseudo-irreversible inhibition.IC50 value of 2.5 ± 0.6 µM . Chrysin also showed the lowest inhibition constant—iK value. The inhibition constants of individual flavonoids were tested using nifedipine as a marker substrate, and for acacetin, apigenin, chrysin, and pinocembrin, the following iK values were determined: 12.1 ± 5.6, 20.2 ± 12.7, 2.4 ± 1.0, and 5.1 ± 1.6 μM, respectively. The corresponding inactivation rate constants were: 0.10 ± 0.02 min−1, 0.11 ± 0.04 min−1, 0.07 ± 0.01 min−1, and 0.04 ± 0.01 min−1, respectively. The inactivation efficiency was determined for each flavonoid as the ratio of the inactivation rate constant and the inhibition constant. Out of all the tested flavonoids, chrysin had the highest inhibition efficiency, which was 0.029 min−1 μM−1  showed a decrease in heme concentration . The assAll flavonoids reduced the heme concentration in the assay with and without the addition of SOD and CAT. The residual heme concentration after incubation with acacetin was 48.88%, and, on retesting with the addition of SOD and CAT, was 63.33%. The residual heme concentration after incubation with apigenin was 45.05%, and, on repeated testing with the addition of SOD and CAT, was 55.11%. The residual heme concentration after incubation with chrysin was 2.9%, while after incubation with the addition of SOD and CAT was 5.5%. The second largest reduction of heme concentration was observed in incubations with pinocembrin, which were at 25.3% and 35.5%, without and with the addition of SOD and CAT, respectively .These results indicate that the inactivation of the cytochrome P450 3A4 enzyme is achieved through the adduct formation of reactive flavonoid intermediate with the heme portion of the enzyme. As the results were confirmed with the use of CAT and SOD, it can be concluded that heme destruction was not caused by reactive oxygen species generated in non-productive cycles of cytochrome P450.p ≤ 0.05).In the pseudo-irreversible inhibition assay, samples were incubated with flavonoids and treated with oxidant, after which they were subjected to dialysis. In the case of pseudo-irreversible inhibition, the enzyme activity should be recuperated. In the incubations with all of the tested flavonoids, there was a significant inhibition of enzyme activity after dialysis with and without treatment with an oxidant . In all As in the previous experiments, the highest reduction of enzyme activity was observed when chrysin was used as an inhibitor. The residual activity of the enzyme after incubation and dialysis with chrysin was 0.57%, and after incubation and dialysis with prior treatment with potassium hexacyanoferrate, it was 1.31% .In case of direct (reversible) inhibition, this type of experiment would show enzyme activity recovery after dialysis. In case of pseudo-irreversible inhibition, enzyme activity would be recovered after treatment with an oxidant and dialysis. As these were not the case with any of the analyzed flavonoids, it can be concluded that all flavonoids show irreversible inhibition of cytochrome P450 3A4.−1 μM−1 vs. 0.029 min−1 μM−1. The observed similarities in the inactivation kinetics are in accordance with literature data confirming that nifedipine and testosterone have different binding sites that overlap [It has been shown that some flavonoids can inhibit the activity of the cytochrome P450 enzymes. Šarić-Mustapić et al.  and Kond overlap . CytochrIC50 and iK values. The molecular docking study of flavonoids binding to cytochrome P450 3A4 has shown a higher binding affinity of chrysin by exposing the B ring to the iron in the active center of the enzyme [The clinical implications of reversible inhibitors  can be avoided if the inhibitor is discontinued from the treatment. More important interactions are those that are irreversible, whereas simply discontinuing the use of the inhibitor will not resolve the interaction issues. In this study, chrysin showed the highest inhibition potential, with the lowest e enzyme . Structue enzyme . Non-submax) of nitrofurantoin in rats [max of these medications. Chrysin is abundant in propolis (28 g/L), and it is the third main flavonoid in honey 5.3 mg/kg [The importance of these described enzyme kinetics can be observed in the context of food–drug interactions, where the administration of chrysin (20–100 µM) significantly increased the AUC and peak serum concentration . Pinocembrin is mostly found in foods such as honey and beverages (tea and red wine), presenting a rich source of this flavonoid [Pinocembrin, a proven irreversible inhibitor of the CYP3A4 enzyme, showed the lowest lavonoid . AccordiIC50 value than chrysin and five times higher iK value, while maintaining similar inactk values as other flavonoids, indicating a lower inhibitory efficacy. Acacetin can mainly be found in plant species such as Turnera diffusa, Robinia pseudoacacia, and Betula pendula [IC50 value and the highest iK value among the four tested flavonoids. The inhibitory potential of apigenin was confirmed in vivo when the combination treatment of apigenin and paclitaxel led to the increase in oral bioavailability of paclitaxel, which was mainly attributed to enhanced absorption in the gastrointestinal tract through the inhibition of P-glycoprotein and reduced first-pass metabolism of paclitaxel through the inhibition of the CYP3A subfamily in the small intestine and/or in the liver by apigenin [Acacetin showed three times higher  pendula , but cer pendula . The parapigenin . Apigeniapigenin .The reversible inhibition of cytochromes P450 3A4 is related to the first step of the cytochrome P450 catalytic cycle . Most ofHowever, as we have previously shown ,29 and cAs cytochromes P450 are hemoproteins, we have studied the binding of flavonoids to the ferrous form of the active site and the binding of a possible reactive intermediate to the heme . To asseThe first step in the catalytic cycle of the cytochrome P450 enzyme is the binding of a substrate to a ferric ion. If the inhibitor shows competitive inhibition to the substate, the enzyme activity can be retrieved by dialysis, as the elimination of the inhibitor enables the complete recovery of the enzyme activity . However5. The results for chrysin indicate that a reactive intermediate generated by cytochrome P450 reacted with heme from cytochrome P450 as well as cytochrome b5.It should be noted that chrysin caused nearly a complete reduction of heme in the incubation: 97.1% without and 94.5% with SOD and CAT ; while tFurther LC-MS analysis was conducted to determine possible reactive flavonoid intermediates either in a form of heme adduct or trapped using glutathione as a radical scavenger (data not shown). However, we were not able to isolate any of the predicted adducts probably due to the instability of the reactive flavonoid intermediate and/or heme/glutathione adduct. A similar behavior was observed with mibefradil, an antihypertensive, withdrawn from the marker due to drug/drug interactions. Although mibefradil caused the reduction of heme, a reactive intermediate was not established .5 in baculosomes were purchased from Thermo Fisher Scientific . Based on cytochrome P450 carbon monoxide assay [+) were purchased from Sigma-Aldrich. Potassium phosphate (p.a.) and dichloromethane (p.a.) were purchased from Kemika d.d. , formic acid  from Semikem d.o.o. , and methanol used for reagent dissolution and chromatography from Merck KGaA . Ultrapure water was used in the incubation mixtures and chromatography. Potassium dihydrogen phosphate (Kemika d.d.) was used to prepare potassium phosphate buffer pH 7.85; pH was adjusted using sodium hydroxide purchased from Semikem d.o.o. Nifedipine (Sigma-Aldrich) and oxidized nifedipine  were used in the study of residual enzyme activity, as well as in inactivation kinetics. Testosterone (Sigma-Aldrich) and 6β-hydroxytestosterone  were used for assays in pseudo-irreversible inhibition. Troleandomycin and diltiazem were obtained from the Agency for Medicinal Products and Medical Devices . Pyridine (p.a.) (Semikem d.o.o.), bovine hemin (Sigma-Aldrich), and dimethylsulfoxide (Semikem d.o.o.) were used in the hemochromopyridine assay. Potassium hexacyanoferrate  was used in the study of reversible and pseudo-reversible inhibition. Superoxide dismutase (SOD) (Sigma-Aldrich), catalase (CAT) (Sigma-Aldrich), and hydrochloric acid  (Semikem d.o.o.) were used in the binding specificity assay. Enzyme incubations were performed in a water bath . Assay of residual enzyme activity and flavonoid inactivation kinetics were performed using high performance liquid chromatography coupled with UV-Vis detection . Residual activity calculations and inhibition kinetics parameters were made using the program R  and Microsoft Excel .Four flavonoids  were used in this study . Recombinant cytochromes P450 3A4 coexpressed with nicotinamide-adenine-dinucleotide phosphate (NADPH) reductase and cytochrome bde assay , the con+: 1000 IU/mL G6PDH = 50:25:1 (v/v/v) was prepared immediately before use. This generating system contains glucose-6-phosphate dehydrogenase that regenerates NADP+ into NADPH, keeping the concentration of the cytochrome P450 coenzyme (NADPH) constant in the incubation. The addition of the generating system  started the reaction. Nifedipine was used to test out the residual enzyme activity at a final concentration of 200 μM. The reaction was stopped using 1 mL of ice-cold 1% solution of formic acid in dichloromethane. The samples were mixed, then centrifuged for 10 min on 1900g . After centrifugation, two layers were formed (water and organic layer). 850 µL of the organic layer was transferred to cuvettes and evaporated. The analyte was then dissolved in 30 µL of methanol. An Agilent Zorbax SB C18 column  was used to analyze the samples by HPLC. The mobile phase was composed of methanol and water in a ratio of 64:36. The analysis was isocratic. The flow was set at 1.0 mL min−1. The injection volume was set at 10 µL. Nifedipine was used as a marker substrate. The reaction observed was the oxidation of nifedipine. Chromatograms were recorded at 254 nm. The duration of the analysis was set at 25 min. The retention time of nifedipine is 7.1 min and that of oxidized nifedipine is 10.8 min [IC50), constant of inhibition (iK), constant of inactivation speed (inactk), and inactivation efficacy (inactk/iK) were determined. Troleandomycin was used as a positive control—irreversible inhibitor of cytochrome P450 3A4. 25 μM concentration of troleandomycin reduced enzyme activity to 35 ± 5%.Enzyme incubations were performed in triplicate, with mechanical stirring in a water bath at 37 °C. Aliquots of flavonoids with a final concentration of 1 μM were dissolved in methanol, transferred to glass tubes, and evaporated to dryness, except in control samples without the inhibitor (flavonoid). After the evaporation of the solvent, an incubation mixture was prepared to a volume of 100 μL, consisting of 5 pmol CYP3A4 enzyme, 50 mM potassium phosphate buffer (pH 7.4), and ultrapure water. An NADPH generating system composed of 0.1 M G6P: 10 mg/mL NADP10.8 min . In bothThe hemochromopyridine assay was used to assess possible heme destruction by reactive intermediate form in cytochrome P450 cycle and was performed according to the method described by Flink and Watson  and Paul2+), by assessing a recovery of enzyme activity after dialysis with or without the addition of an oxidant. Three types of experiments were performed for each flavonoid: incubation mixture without flavonoids (control), incubation mixture with flavonoid, and incubation mixture with flavonoid to which oxidant was added after incubation. Each incubation with the inhibitor was performed for 30 min using the same experimental settings as described above  and Microsoft Excel .A one-sided IC50, iK, inactk values and the highest inactivation efficacy. All flavonoids reduced the heme concentration of the enzyme, confirming that this is an irreversible inhibition by reactive intermediate that cannot be prevented by the addition of SOD and CAT. None of the tested flavonoids act as reversible or pseudo-irreversible inhibitors of the CYP3A4 enzyme at 25 μM concentration, as the enzyme activity could not be recovered with dialysis with and without the addition of potassium hexacyanoferrate. As these flavonoids can abundantly be found in various foods such as fruits, vegetables, spices, teas, and red wine, there is a possibility that they can interfere with various xenobiotics that are CYP3A4 substrates. Further in vivo studies are needed to completely investigate the possibilities of such food–drug interactions, as well as the possible contribution of other enzymes and transporters in the interactions.Acacetin, apigenin, chrysin, and pinocembrin inhibit the CYP3A4 enzyme activity in vitro. Chrysin is the most potent enzyme inhibitor, with the lowest


To assess the hypofluorescent foci (HFF) on late-phase indocyanine green angiography (ICGA) in central serous chorioretinopathy (CSC) using short-wavelength fundus autofluorescence (SW-FAF), near-infrared autofluorescence (NIR-AF), and fluorescein angiography (FA). The HFF area on late-phase ICGA for at least 20 min was compared with the area of abnormal foci on SW-FAF, NIR-AF, and FA. In 14 consecutive patients , four kinds of images of 27 eyes were acquired. The mean age ± standard deviation (range) was 46 ± 9.2 years (31–69 years). The HFF on late-phase ICGA were found in 23 eyes . From the results of simple regression analysis, we obtained the following three formulas. The HFF area on ICGA = 1.058 × [abnormal SW-FAF area] + 0.135, the HFF area on ICGA = 1.001 × [abnormal NIR-AF area] + 0.015, and the HFF area on ICGA = 1.089 × [abnormal FA area] + 0.135. Compared to SW-FAF and FA, NIR-AF was found to be the easiest method to detect the HFF on late-phase ICGA, which may indicate melanin abnormalities, especially a decrease, in the retinal pigment epithelium. Central serous chorioretinopathy (CSC) is a disease that most commonly occurs in men 9.9/100,000), mainly in their 30s to 50s, and its incidence is six times higher than that in women 1.7/100,000). It causes localized serous retinal detachment (SRD) in the macula 00,000, m. Even in/100,000.The retinal pigment epithelium (RPE) contains melanin, melanolipofuscin, and lipofuscin . StudiesIt is reported that fluorescence in the RPE over the drusen is less intense than that of the adjacent RPE on ICGA . Chang eThe appearance of HFF on late-phase ICGA has the potential to show changes in pathology in vivo, but the testing is laborious and invasive. The changes around the RPE should be visible in other modalities. In this study, we comprehensively assessed the HFF on late-phase ICGA imaging by using SW-FAF, NIR-AF, and FA.This study was conducted at the Department of Ophthalmology, Keio University Hospital, Tokyo, Japan, between October 2019 and March 2020. Data of all consecutive patients with CSC who underwent consultation during this period were prospectively assessed. All procedures adhered to the tenets of the Declaration of Helsinki. Written informed consent was obtained from all patients before they underwent angiography and other examinations. This study was approved by the Keio University School of Medicine Ethics Committee .The eligibility criterion was CSC in either eye. The exclusion criteria were as follows: patients with drusen within 30 degrees, drusenoid or hemorrhagic pigment epithelium detachment, high myopia (<−6.0 diopters), dome-shaped macula, vitreomacular traction syndrome, eyes with choroidal neovascularization detected by OCT angiography, and any history of uveitis or other retinal diseases.We obtained images using SPECTRALIS HRA+OCT2; Heidelberg Engineering, Heidelberg, Germany. Images created by using SW-FAF, NIR-AF, FA, and ICGA for at least 20 min were analyzed. SW-FAF images were acquired at an excitation wavelength of 488 nm and an emission spectrum over 500 nm, and NIR-AF images were acquired at an excitation wavelength of 787 nm and an emission spectrum over 820 nm .For the data processing, we used Microsoft PowerPoint 2019 (version 2102) and ImageJ (version 1.52a), which is an open-source image processing and analysis program written in Java by Wayne Rasband, National Institutes of Health, Bethesda, MD, USA. In 14 consecutive patients , four kinds of images  of 28 eyes were acquired. Images of each eye were obtained on the same day. The HFF on late-phase ICGA were defined as clearly hypofluorescent compared to iso-fluorescent on late-phase ICGA. In chronic CSC, HFF may sometimes include hyperfluorescent foci, which was also defined as HFF. On the PowerPoint, we drew areas containing HFF on late-phase ICGA images using the ‘Curve’, and we then colored those HFF areas. If there were many abnormal HFF, the top biggest 3 abnormal foci on late-phase ICGA were measured. To investigate how many HFF obtained on late-ICGA could be picked up by the other three types of images , we similarly selected abnormal foci that matched the HFF of the ICGA. After confirming that all image ratios matched, we saved the image under another file name.We then used ImageJ and selected the ‘Wand (tracing) tool’, ‘Analyze’, and ‘Measure’. Since abnormal area sizes were shown on the screen, we converted the real size from the ratio of each image. We repeated the same steps for processing each image, and the HFF areas on late-phase ICGA were compared with the area of abnormal foci on SW-FAF, NIR-AF, and FA images, which corresponded to the HFF on late-phase ICGA images.Simple regression analyses were performed using IBM SPSS Statistics for Windows, Version 27.0 .In 14 consecutive patients , 28 eyes were analyzed. The mean age ± standard deviation (range) was 46 ± 9.2 years (31–69 years), and the mean symptom duration was 30.0 ± 72.9 months (0.5–288 months). The mean subfoveal choroidal thickness was 417.2 ± 103.5 µm (265–624 µm), and the mean axial length was 24.1 ± 1.0 mm (22.3–25.9 mm). Only one patient had a history of prior/ongoing corticosteroid use.2 (0.03–26.5 mm2) (n = 23). The mean abnormal foci area on SW-FAF was 3.5 ± 6.5 mm2 (0.03–26.4 mm2) (n = 23). The mean abnormal foci area on NIR-AF was 3.8 ± 6.9 mm2 (0.03–26.5 mm2) (n = 23). The mean abnormal foci area on FA was 3.4 ± 6.2 mm2 (0.01–26.3 mm2) (n = 23).There were four patients with unilateral CSC, where the contralateral unaffected eye had no HFF on late-phase ICGA. In addition, one eye showed quiescent choroidal neovascularization using OCT angiography in the contralateral eye of CSC. Therefore, we excluded five eyes from the HFF analysis. The mean HFF area size on late-phase ICGA over 20 min was 3.9 ± 6.9 mm2) and the size of abnormal area on SW-FAF (mm2), the size of abnormal area on FA (mm2), and the size of abnormal area on NIR-AF (mm2) (AF (mm2) . From the results of simple regression analysis, we obtained the following three formulas. The HFF area on ICGA = 1.058 × [abnormal SW-FAF area] + 0.135, the HFF area on ICGA = 1.001 × [abnormal NIR-AF area] + 0.015, and the HFF area on ICGA = 1.089 × [abnormal FA area] + 0.135. Compared to SW-FAF and FA, NIR-AF was found to be the easiest method to detect the HFF found in the late-phase of ICGA.In this study, we could detect any abnormal foci on SW-FAF, NIR-AF, and FA within the HFF area on late-phase ICGA. In particular, it was found that NIR-AF easily detected the HFF seen in the late-ICGA visually . AlthougIn ICGA, we can observe the course of the dye of ICG changing in vivo from the early phase to the late phase. It is reported that the fluorescence of the RPE over the surface of drusen in the eye of a monkey by using ICG was less intense than that of the adjacent RPE . DrawingThe NIR-AF signal is thought to originate from melanin and melanolipofuscin ,10,11. FAlthough SW-FAF and FA images are obtained by short wavelength, the findings seen on the two modalities are different using fluorescein dye. The SW-FAF signal is thought to originate from lipofuscin and melanolipofuscin ,10,11. IAs the strengths of our research, what we see in NIR-AF and ICGA may not necessarily be the same, but we were able to obtain overlapping findings in many cases. In our previous study, the HFF on late-phase ICGA was thought to correspond to lipid accumulation at Bruch’s membrane to the choriocapillaris level , and theThe limitations of this study are as follows. Firstly, we wanted to analyze our data by multiple regression analysis using the data of the duration of subjective symptoms or gender differences. However, it was not possible to determine when the HFF occurred in the contralateral eye. Moreover, we had only two unilateral eyes in women. Therefore, we performed a simple regression analysis instead of multiple regression analysis. The number of patients was too small, and the symptom duration was too broad in this study. We need to obtain follow-up data on many patients in the future study. In addition, we would like to investigate these HFF using deep learning to check the replication in the future study.In this study, we could detect any abnormal foci with high probability in SW-FAF, NIR-AF, and FA within the HFF area on late-phase ICGA, and we found NIR-AF easily detected the HFF seen in the late-ICGA visually. FA and ICGA testing are laborious and invasive, but SW-FAF and NIR-AF require short image acquisition time. NIR-AF is the least dazzling and has the shortest image acquisition time than the other modalities, which may easily detect those overlapped findings of RPE disorder and lipids at Bruch’s membrane. It will be interesting to clarify whether the HFF in NIR-AF is a significant risk factor for SRD in the future study. If we prove that, NIR-AF might be able to replace late ICGA images when we evaluate RPE disorders and SRD development.


Cue-induced drug craving and disinhibition are two essential components of continued drug use and relapse in substance use disorders. While these phenomena develop and interact across time, the temporal dynamics of their underlying neural activity remain under-investigated. To explore these dynamics, an analysis of time-varying activation was applied to fMRI data from 62 men with methamphetamine use disorder in their first weeks of recovery in an abstinence-based treatment program. Using a mixed block-event, factorial cue-reactivity/Go-NoGo task and a sliding window across the task duration, dynamically-activated regions were identified in three linear mixed effects models (LMEs). Habituation to drug cues across time was observed in the superior temporal gyri, amygdalae, left hippocampus, and right precuneus, while response inhibition was associated with the sensitization of temporally-dynamic activations across many regions of the inhibitory frontoparietal network. Methamphetamine-related response inhibition was associated with temporally-dynamic activity in the parahippocampal gyri and right precuneus , which show a declining cue-reactivity contrast and an increasing response inhibition contrast. Overall, the declining craving-related activations (habituation) and increasing inhibition-associated activations (sensitization) during the task duration suggest the gradual recruitment of response inhibitory processes and a concurrent habituation to drug cues in areas with temporally-dynamic methamphetamine-related response inhibition. Furthermore, temporally dynamic cue-reactivity and response inhibition were correlated with behavioral and clinical measures such as the severity of methamphetamine use and craving, impulsivity and inhibitory task performance. This exploratory study demonstrates the time-variance of the neural activations undergirding cue-reactivity, response inhibition, and response inhibition during exposure to drug cues, and suggests a method to assess this dynamic interplay. Analyses that can capture temporal fluctuations in the neural substrates of drug cue-reactivity and response inhibition may prove useful for biomarker development by revealing the rate and pattern of sensitization and habituation processes, and may inform mixed cue-exposure intervention paradigms which could promote habituation to drug cues and sensitization in inhibitory control regions. This potential crisis is compounded by the fact that despite decades of research, assessments of MUD are still largely reliant on clinical interviews5 and data on effective interventions for MUD remains inconsistent6, with growing calls to better delineate the neurobiology of MUD to identify novel treatment targets and clinically-relevant biomarkers7. In tandem with research elucidating the involvement of a plethora of cognitive functions in the MUD8, functional magnetic resonance imaging (fMRI) studies on the neurobiology of MUD have characterized several functional brain changes associated with cognitive alterations in the MUD, methamphetamine craving, use history and relapse risk, and treatment outcomes10.The prevalence and health burden of methamphetamine use disorder (MUD) continues to increase globally12, and failures of executive control and response inhibition13. These phenomena widely figure in models of a substance use disorder, such as the “impaired response inhibition and salience attribution” model14 and dual-process models, which comprise automatic approach behavior towards substances and reduced abilities to inhibit these behaviors15. An increasing number of task-based fMRI studies in MUD have investigated the neural correlates of either methamphetamine cue-reactivity, using conventional cue exposure tasks19 or cognitive control and response inhibition, using varieties of Stroop, Stop Signal or Go-NoGo tasks22, but an approach with greater ecological validity has been to assess response inhibition concurrently with cue-reactivity. These latter studies are motivated by the interrelationship between response inhibition and cue-reactivity, with drug cue exposure hampering inhibitory control25 and poor inhibitory control precipitating higher induced craving26. This line of fMRI research typically involves mixed tasks to investigate the response inhibition in the context of exposure to drug cues, such as Go-NoGo tasks in which Go and NoGo signals are independently mixed through neutral and substance cues28 or in which substance cues are themselves the NoGo signal30.Two central aspects of methamphetamine use disorder are a characteristic reactivity to drug cues 34 which might be explained through extinction learning35, though regional sensitization to pictures of angry faces has also been observed36. Considering the potentially multiphasic nature of the cue-reactivity process which unfolds over seconds and minutes and primarily involves different regions and networks at each stage11, it’s not surprising that three recent studies on the MUD and opioid use disorder have found evidence of temporally dynamic activation patterns during cue-reactivity in regions such as the amygdala, the dorsal anterior cingulate cortex, the ventromedial prefrontal cortex, the ventral striatum, the caudate nuclei and insular cortices, and various prefrontal regions39. Given the temporally dynamic involvement of regions such as the bilateral motor and prefrontal cortices in response inhibition40 and the dynamic reconfiguration of functional brain networks41, it’s reasonable to expect similarly dynamic activation patterns to be implicated in successful and dysfunctional response inhibition in individuals with substance use disorders.A notable characteristic of the above-mentioned studies is an assumption of time-invariant voxel-wise or regional activation, whereby in both response inhibition and cue-reactivity tasks average responses are obtained across the entire task duration. This conventional “static” approach might be problematic in light of the evidence that activation patterns during exposure to emotionally salient stimuli are often dynamic and vary across the task duration, for example when brain regions demonstrate various patterns of habituation to emotionally negative cues or reward stimuliUsing data obtained from the first fMRI implementation of a novel mixed cue-reactivity/Go-NoGo task, this study aims to investigate temporally dynamic brain activation patterns that underlies methamphetamine cue-reactivity, response inhibition, and methamphetamine-related response inhibition (response inhibition during exposure to methamphetamine cues) in individuals with MUD. These activation slopes may reflect the sensitization (positive slope) or habituation (negative slope) of brain regions as they engage in response inhibition and the processing of methamphetamine cues. Further, the correlation of activation slopes with clinical and behavioral variables will be investigated, providing an initial estimate of the potential clinical utility of activation slopes.42, (2) abstinence from any substance for at least one week, with the exception of nicotine, based on self-report and confirmed by urine drug screening, (3) right-handedness, determined using the Edinburgh Handedness Inventory43, and (4) age between 20 and 40 years. Exclusion Criteria were (a) any comorbid axis‐I disorders other than drug dependence, based on DSM-IV TR criteria, (b) ineligibility for MRI scanning , (c) history of head trauma resulting in neurological disorders. Nine participants were excluded from fMRI analyses due to excessive movement during scanning, leaving 53 individuals (see “Sixty-two men with MUD age 32.12 ± 5.89) were recruited from addiction treatment centers in Tehran, Iran. Inclusion criteria were (1) Diagnosis of methamphetamine dependence (for at least 6 months) according to the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria (DSM-IV TR)2 ± 5.89 The research protocol was designed and implemented in accordance with the Declaration of Helsinki. After a referral from treatment centers to the research team, individuals were informed regarding the aims of the project, the collected information and measures are taken to ensure anonymity, scanning procedures, the fMRI task and its potential to induce methamphetamine craving, and that they can exit the study at any point with no implications for their ongoing treatment. After the consent form was read both out loud by a psychologist and by the individual to ensure comprehension, participants provided written, informed consent prior to further screening for enrollment. The data collected from each participant was sent to the primary data analyst and anonymized before further processing. The study protocol was reviewed and approved by the ethical review board of the Tehran University of Medical Sciences with the approval code 93-02-98-23869.44, the Depression Anxiety and Stress Scale-21 (DASS-21)45, and the Positive and Negative Affect Schedule (PANAS)46. The number of days on which participants abused methamphetamine, cannabis, alcohol, sedatives, hallucinogens , cocaine, hard opioids (heroin and crack heroin) and soft opioids  in the last 30 days before the start of treatment was assessed using a Drug Abuse Profile instrument. A Risky Behaviors Profile instrument was utilized to assess the history of injecting drug use, high-risk sexual behavior, incarceration, drug selling, and physical altercation.Participants were abstinent prior to scanning, but were allowed to smoke. After arriving at the imaging center, participants were interviewed by two clinical psychologists, and several measures were administered prior to scanning. Collected data included demographic information, mental status examination, the Barratt Impulsiveness Scale-11 (BIS-11)Methamphetamine craving was assessed using a 0–100 Visual Analog Scale (VAS) before MR scanning, with the question “how much craving are you experiencing right now?”. After scanning, participants were again assessed with PANAS and rated their craving , neutral images, negative emotional cues, or methamphetamine-related cues. Each block contained 18 Go signs  and 6 NoGo signs (circles). Each stimulus lasted one second and was followed by a jittered inter-stimulus interval generated using a gamma probability density function (mean = 0.5)  scanner in the Tehran University of Medical Sciences, Iran. Functional scans were collected using a 2D gradient echo EPI sequence, and each volume was comprised of 40 contiguous axial slices  = 192 × 192, in-plane voxel size 3.0 mm × 3.0 mm, slice thickness 3 mm, FA = 90°). The scanning session lasted 806 s. A high-resolution T1-weighted structural image was also acquired for each participant for co-registration during preprocessing and to exclude participants with any structural abnormality. Structural images were acquired through a sagittal T1-weighted magnetization-prepared rapid acquisition (MP-RAGE) sequence with the following parameters: repetition time = 1800 ms, echo time = 3.44 ms, FOV = 256 cm × 256 cm, flip angle = 7°, 1 mmwww.fmrib.ox.ac.uk/fsl) version 6.0.3 was used to preprocess structural and functional data49. Structural data was skull-stripped to remove non-brain tissue from the structural T1-weighted images using the Brain Extraction Tool (BET). BET parameters were chosen based on each individual skull size.FSL  of functional images to the high-resolution T1 images, nonlinear registration of the T1 images to the standard Montreal Neurological Institute (MNI) space with 12 degrees of freedom, intensity normalization, smoothing with a 5-mm full-width at half-maximum (FWHM) Gaussian kernel, denoising with melodic ICA, high-pass temporal filtering .Functional data were analyzed using the fMRI Expert Analysis Tool (FEAT), part of FMRIB’s Software Library. The functional pre-processing included the removal of the first five volumes, motion correction with 6 degrees of freedom, interleaved slice-timing correction, linear Boundary-Based Registration  analysis. “High movement subjects” were defined as those with displacement > 4 mm and also DVARS > 75 in more than ten volumes in a single block (36 s), and were excluded from the analyses (9 subjects).High motion effects on fMRI time series were identified using the DVARS metricThe data used in this study have been analyzed in a conventional GLM framework before. .51. First, the whole brain was parcellated into 246 regions based on the BNA. The BNA masks in MNI space were then registered to each subject’s space using the transformation matrices derived from the pre-processing step and after determining subject-specific masks for each ROI across the 53 subjects, the mean activations and standard errors were calculated. We then used a sliding window over each subject’s fMRI time series, separating the task duration into three overlapping windows with a window duration of two task runs (396 s) and a sliding interval equal to one run (198 s) , Neutral Successful Go (NSG), Drug Successful NoGo (DSNG) and Drug Successful Go (DSG) events DSNG + DSG), Neutral (NSNG + NSG), NoGo (DSNG + NSNG), Go (DSG + NSG), response inhibition during methamphetamine cue exposure (DSNG − DSG) and response inhibition during neutral cue exposure (NSNG − NSG). For each of the three pairs of conditions, “dynamically-activated brain regions” were defined as those regions in which the activations associated with the two conditions across time have differing slopes. These regions were identified on the group level by fitting three Linear Mixed Effects (LME) models to subject beta coefficients in each region in R, version 3.6.253 , response inhibition conditions , and methamphetamine-related response inhibition conditions . Using three GLM models, beta coefficients for each condition (each one of the two conditions in a contrast) in each window were estimated separately Fig. e. Overal253 Fig. e. The thp < 0.001. Since a significant interaction term in an LME model indicates an intersection of the lines associated with different levels of one variable drawn across the levels of the other variable, observing a significant interaction of condition and time variables in a brain region in the LME models would mean two things: firstly, that the neural activity associated with one or both conditions is temporally evolving across the three windows (since two flat activation lines would not intersect) and secondly, that the evolution of neural activity differs between the two conditions in the model. Accordingly, it could be said that regions with a significant condition-by-time interaction in the first LME show “temporally dynamic cue-reactivity”, regions with a significant condition-by-time interaction in the second LME show “temporally dynamic response inhibition, and regions with a significant condition-by-time interaction in the third LME show “temporally dynamic methamphetamine-related response inhibition”. For each model, the temporal evolution of activity associated with either condition was examined by plotting the beta values (those from the GLM models in each window) for the relevant conditions across the three windows.To guard against false positives and given the number of tests performed, regions with significant main effects of time and condition and those with a significant condition-by-time interaction effect were extracted after a False Discovery Rate (FDR) correction with a threshold of After the identification of regions with dynamic activity in the group-level analysis, the relationship between the strength of this dynamic activity for each pair of conditions and behavioral and clinical data was investigated. For every ROI exhibiting a significant condition-by-time interaction in each model, separate linear models were fit for each subject with the form:p < 0.05 were used for these tests.For each dynamically activated region in one of the three LMEs, individuals with positive DSNG + DSG vs. NSNG + NSG) was significant in the medial superior frontal gyrus, sensory thalamus, paracentral lobule, superior temporal gyrus, rostroventral inferior parietal lobule, post-central gyrus, hypergranular insula, right caudodorsal cingulate gyrus, medio-ventral occipital cortex, and left sensory thalamus  and bilateral caudal parahippocampal gyri , right ), right rostroposterior superior temporal sulcus  and bilateral medial precunei , right )  in the cue-reactivity LME is significant in the right lateral superior temporal gyrus , bilateral rostral superior temporal gyri  and right inferior temporal gyrus , the left precuneus , the right medial  and lateral amygdala , and the right rostral hippocampus   but all except left rostral superior temporal gyrus and right medial amygdala show a significant negative main effects of time  to drug cues compared to neutral cues Fig. .Figure 3(DSNG + NSNG vs. DSG + NSG) , right ), right medial precuneus , medio-ventral and lateral occipital cortices  , right ), right cingulate gyrus , left insula , right angular gyrus , right middle temporal gyrus  and left supramarginal gyrus  showed falling inhibitory activations (habituation) to NoGo cues, while the bilateral precentral gyri , right ), right superior parietal lobule , and right opercular inferior frontal gyrus ) showed increasing contrasts (sensitization)  is highly significant across much of the frontal cortex, superior temporal gyrus, inferior parietal lobule, precuneus, post-central gyrus, dorsal insula, medioventral occipital cortex, and basal ganglia   with an attenuating inhibitory response while viewing drug-related cues (DSNG − DSG) but not during neutral cue exposure (NSNG − NSG). The main effects of condition and time are not significant in this region individually. Bilateral medial parahippocampal gyri also show significant condition-by-time interactions , left: ) , left: ), unlike the main effect of time ) Fig. c, and hame Table .Figure 5(DSNG + DSG > NSNG + NSG) and to methamphetamine-related response inhibition (DSNG − DSG > NSNG − NSG), but increasing values of the response inhibition contrast (DSNG + NSNG > DSG + NSG) across time. In the right precuneus as well, the cue-reactivity and methamphetamine-related response inhibition contrasts decrease in value over time but the response inhibition contrast remains mostly stable.For the three ROIs with a dynamic involvement in methamphetamine-related response inhibition, values of three contrasts for cue-reactivity, response inhibition and methamphetamine-related response inhibition were estimated across the overlapping windows for further exploration, and the results are illustrated in Fig. p-values = 0.017 and 0.028, respectively) and shorter average reaction times  compared to those with negative activation slopes. Having a positive cue-reactivity slope in the right superior temporal gyrus was associated with greater methamphetamine use  and higher baseline craving , but shorter average reaction times on the task . Having a positive cue-reactivity slope in the left precuneus was associated with the amount of money spent procuring methamphetamine  but lower motor impulsivity , and a positive slope in the right amygdala was associated with fewer risky behaviours . Considering the response inhibition linear models, Individuals with positive activation slopes in the right superior temporal gyrus reported higher motor  and total impulsivity  and more money spent procuring methamphetamine , but a lower craving after the task . Having a positive response inhibition slope was associated with a longer average reaction time in the left parahippocampus , but a shorter average reaction time in the right parahippocampus  . There have been reports of prefrontal sensitization to salient cues36, and it has been observed that the prefrontal cortex is implicated in the dysfunctional behavioral regulation seen in the MUD during response control tasks22. The observation of dynamic activity in prefrontal regions was expected, given their involvement in inhibitory control networks51 and response inhibition in substance use disorders64.More than a hundred regions in our LME model showed dynamic response inhibitory activity. This may not be surprising, as response inhibition is associated with large-scale neural activity54 had dynamic activation patterns in this study. Notably, while dynamic cue-reactivity was associated with a generalized habituation effect, these regions showed two broad temporal activation patterns. The middle temporal gyrus, the left insula, the right cingulate gyrus, the right middle temporal gyrus, and supramarginal gyrus showed falling inhibitory activations while the precentral gyrus, left superior parietal lobule, and opercular frontal gyrus showed increasing activations (sensitization). This might reflect differences in response inhibitory processes that these regions contribute to, such as error monitoring and attentional control66, or the involvement of these regions in other networks that interact with the response inhibition network in individuals with substance use disorders, such as the insula in the salience network or the middle frontal gyrus in self-directed processing14. Compared to the cue-reactivity contrast, increasing response-inhibition-associated activity showed more mixed associations with behavioral variables in t-test comparisons with individuals who had decreasing activation across a number of regions. An increasing response-inhibition contrast in the right superior temporal gyrus was associated with both higher impulsivity and higher drug use but with a lower craving after the task, while parahippocampal sensitization to response inhibition was associated with both shorter and longer reaction times in the left and right parahippocampi, respectively. These contradictory associations may indicate that the dynamic neural correlates of response inhibition are reliable indicators of neither addiction severity nor impulsivity and inhibitory capacity, though they may also have resulted from unnoticed design flaws. Turning to regression analyses without dichotomization, amount of money spent procuring methamphetamine is correlated with the interaction β coefficient in the right lateral superior temporal gyrus after FDR correction.Most of the regions involved in response inhibition in a recent meta-analysis of Go-NoGo tasks68, and based on the response inhibition literature, dopaminergic inhibition, and network decoupling of precuneal activity may be important for successful response inhibition71. Precuneal involvement in cue-reactivity in substance use disorders might be related to its role in the default mode network and self-referential processing in general14, and, interestingly, it has been argued that the precuneus might be an important node for the integration of contradictory executive control and cue-reactivity processes72. Considering the above, the decreasing activation associated with drug-related inhibition in the right precuneus may reflect a lessening effect of drug cues in hampering response inhibition across the task duration. Since it appears that the response inhibition contrast in the precuneus is mostly stable across time while cue-reactivity and methamphetamine-related response inhibition contrasts decline, habituation to drug cues or top-down suppression of precuneal cue-reactivity, rather than the role of the precuneus in response inhibition per se, maybe the responsible mechanisms.The bilateral parahippocampal gyri and the right precuneus were the only regions with a dynamic methamphetamine-related response inhibition. Several meta-analyses have demonstrated that drug cue-reactivity is associated with heightened precuneal activation73 and increases in its connectivity within the default mode network74, both the right and the left parahippocampus generally show higher activations in response to drug-related cues compared to neutral cues76, and response inhibition-associated parahippocampal dysfunction has been observed in individuals with substance use disorders compared to healthy controls20. As part of the default mode network and given its association with drug cue-reactivity, it was expected that similar to the precuneus, the cue-reactivity contrast in the parahippocampal gyri would decrease, reflecting both habituation processes and task-engagement-related suppression. Some evidence also exists for parahippocampal habituation during exposure to emotionally salient stimuli78 and for the role of the parahippocampus in the extinguishing of drug cue associations79. However, the parahippocampus is also involved in neural networks underlying associational memory and learning81 and might be activated to support learning during response inhibition tasks14. Indeed, increasing parahippocampal recruitment during a learning task has been observed before82. These dual roles of the parahippocampus in cue habituation and learning could explain why the cue-reactivity contrast decreased while the response inhibition contrast increased in the parahippocampal gyri during the task, and is supported by the observation that drug-related inhibition remained mostly stable, while inhibition during neutral cue exposure was associated with increasing parahippocampal activity.The parahippocampus has also been implicated in substance use disorders. Addictive disorders are associated with parahippocampal gray matter changes84, and right lateralization of dynamic response to salient stimuli has been observed in the right amygdala, inferior parietal lobule, and hippocampus85. It has been argued that while the left amygdala is involved in sustained stimulus evaluation, the right amygdala might be more specialized for dynamic stimulus processing34. This may also explain why sensitization to response inhibition in the right parahippocampal gyrus was associated with better response inhibitory performance, but sensitization in the left parahippocampus had the opposite association.An interesting observation in this study was the right-lateralization of dynamically active regions across the three contrasts. Some evidence exists that the right hemisphere may be more important in response inhibitory and attentional control processesIn general, it appears that regions with temporally dynamic methamphetamine-related response inhibition increasingly engage in response-inhibitory processing while developing a habituation to drug cues. This may indicate a general pattern underlying repeated drug cue exposure, with neural resources gradually shifting to inhibit undesirable responses while a habituation to drug cues develops and the initial intensity of cue reactivity dissipates.87. While such issues may be ameliorated by the choice of a blocked presentation of different cue types, the results are likely confounded by these effects. A further concern with our separation of the signal into three windows would be the resulting loss of power; and while we had a moderately sized sample, the reported observations need to be replicated in larger samples. Lastly, we used no measure of craving across the task duration, which would have allowed the analysis of temporal correlations between craving and neural activity, as in one recent study by Murphy et al.38.While the results of this exploratory investigation are promising, several limitations are important to point out. Firstly, we included no healthy control group, and so the specificity of observed patterns to individuals with MUD is unclear. Also, all participants were men, treatment seeking individuals MUD, limiting the generalizability of our observations. Regarding the task design, an inherent limitation introduced by our use of a mixed drug cue and negative emotional Go-NoGo task is the potential carry-over effects of salient cues on brain activity during subsequent blocks88, and it has been recently argued that amygdalar habituation is a more reliable index than mean amplitude89. Dynamic methamphetamine-related response inhibition was observed in regions in which the neural activations associated with cue-reactivity and response inhibition followed broadly opposing slopes across time, namely in the parahippocampal regions and the precuneus, suggesting that these regions may be important hubs where response inhibitory and cue-reactivity processes integrate. Investigating the dynamic interplays of cognitive processes in these regions may help biomarker development and suggest new targets for interventions, and it has been suggested that failures to inhibit precuneal cue-reactivity may predict relapse90, and impairments of parahippocampal habituation are associated with poorer treatment outcomes in cocaine users39. Future studies could make use of better power analyses, flexible sliding window sizes and inference methods, prospective designs, and replication across different populations and time-points to assess the stability, generalizability, and potential predictive utility of these dynamic activation parameters.This study provides preliminary evidence that a mixed event-block Go-NoGo/cue-reactivity task can be used to assess the temporal dynamics of cue-reactivity, response inhibition, and methamphetamine-related response inhibition. The regions with a temporally dynamic response are involved in various neuro-cognitive aspects of addictive disorders. Notably, we observed dynamic amygdalar activity in both response inhibition and cue-reactivity contrasts, and there is extensive literature on the time-variance of amygdalar activitySupplementary Information.


Protein kinases exert physiological functions through phosphorylating their specific substrates; however, the mode of kinase–substrate recognition is not fully understood. Rho-kinase is a Ser/Thr protein kinase that regulates cytoskeletal reorganization through phosphorylating myosin light chain (MLC) and the myosin phosphatase targeting subunit 1 (MYPT1) of MLC phosphatase (MLCP) and is involved in various diseases, due to its aberrant cellular contraction, morphology, and movement. Despite the importance of the prediction and identification of substrates and phosphorylation sites, understanding of the precise regularity in phosphorylation preference of Rho-kinase remains far from satisfactory. Here we analyzed the Rho-kinase–MYPT1 interaction, to understand the mode of Rho-kinase substrate recognition and found that the three short regions of MYPT1 close to phosphorylation sites (referred to as docking motifs (DMs); DM1 (DLQEAEKTIGRS), DM2 (KSQPKSIRERRRPR), and DM3 (RKARSRQAR)) are important for interactions with Rho-kinase. The phosphorylation levels of MYPT1 without DMs were reduced, and the effects were limited to the neighboring phosphorylation sites. We further demonstrated that the combination of pseudosubstrate (PS) and DM of MYPT1 (PS1 + DM3 and PS2 + DM2) serves as a potent inhibitor of Rho-kinase. The present information will be useful in identifying new substrates and developing selective Rho-kinase inhibitors. Eukaryotic protein kinases are a large family of highly conserved enzymes mediating the phosphorylation of substrate proteins in Ser/Thr/Tyr residues and they play key roles in the cellular signal transduction pathway. The activity of each protein kinase is temporally and spatially regulated, and aberrant activation or inactivation of kinase sometimes leads to disease. The catalytic domain of kinase specifically forms a complex with the target substrate and ATP. In addition to the catalytic domain, intra- and/or intermolecular domains relating to protein–protein interactions also contribute to the specification of substrates. Most protein kinases recognize the ‘consensus sequence’ surrounding the substrate phosphorylation site through the active site ,2. The cWe have previously developed a novel method  of efficiently screening protein kinase substrates ,8,9. In Rho-associated kinases (ROCKs) ROCK1 and ROCK2/Rho-kinase (we refer to Rho-kinase) are Ser/Thr-protein kinases that regulate cellular cytoskeletal organization downstream of the small GTPase Rho. ROCKs are broadly expressed in tissues, and aberrant activation of ROCKs has been reported to be involved in various diseases, including cardiovascular diseases, neurodegenerative diseases, ocular diseases, and tumors ,11,12. MAnti-GFP monoclonal mouse antibody , anti-MYPT1-pT853 (pT855 in Rat) rabbit polyclonal antibody , anti-MYPT1-pT696 (pT697 in Rat) rabbit polyclonal antibody , and Alexa555-Phalloidin  were purchased. Anti-GST polyclonal rabbit antibody and anti-MYPT1-pT854 polyclonal rabbit antibody were produced against recombinant GST and phosphopeptide CREKRRpSTGVSF, respectively. cDNAs encoding bovine Rho-kinase , rat MYPT1 , human ROCK1 (UniProt Q13464-1), and human MRCKβ  were cloned from cDNA libraries, and subcloned into pEF-BOS-GST , pEGFP-cGST-recombinant kinases were purified from Sf9 cells using a baculovirus expression system . The cel5 cells/well in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum and cultured overnight. The cells were transfected with plasmids using LIPOFECTAMINE 2000TM reagent , according to the manufacture’s protocol. Glutathione Sepharose beads were incubated with COS7 cell lysates transfected with the indicated plasmids in lysis buffer  for 1 h at 4 °C, and the beads were subsequently washed three times with lysis buffer. After washing, the beads were resuspended in SDS–PAGE sample buffer, and the bound proteins were subjected to immunoblot analysis using the indicated antibodies. The signals were visualized using HRP-conjugated secondary antibody and ECL prime detecting reagent  by X-lay film or imaging analyzer LAS 4010 .COS7 cells were seeded on a 6-well dish at a density of 1.8 × 105 cells/well in DMEM with 10% fetal bovine serum and cultured overnight. The cells were transfected with plasmids using LIPOFECTAMINE 2000TM reagent, according to the manufacture’s protocol. After 24 h, the cells were washed twice with PBS, lysed in SDS–PAGE sample buffer and, subjected to immunoblot analysis using the indicated antibodies. The signals were visualized using HRP-conjugated secondary antibody and ECL prime detecting reagent  by X-lay film or imaging analyzer LAS 4010 . Signal intensities of phospho-GFP-MYPT1 and GFP-MYPT1 were analyzed using Fiji software ATP [1–20 GBq/mmol]), GST-Rho-kinase-cat-L or other kinases (1–10 nM), and substrates for 10–20 min at 30 °C. The reaction mixtures were applied to cation-exchange membranes and washed with 75 mM phosphoric acid three times, which was followed by scintillation counting.The kinase reactions were performed in 50 μL of a reaction mixture , according to the manufacture’s protocol. After 24 h, the cells were incubated in fresh DMEM containing 10% calf serum for 20 min, fixed with 3.7% formaldehyde in PBS for 10 min and then treated with PBS containing 0.2% Triton X-100 for 10 min. The fixed cells were blocked with 1% bovine serum albumin for 30 min and stained with the indicated antibodies. Cover slips were mounted with Fluoromount  and the fluorescence was examined using a confocal laser scanning microscope LSM 780 and LSM5 PASCAL built around an Axio Observer Z1 or Axiovert 200M under the control of LSM software . Images were processed using ImageJ or Photoshop .NIH3T3 cells were seeded on polylysine-coated 13-mm coverslips at a density of 4 × 10Prism  was used for statistical analysis. Data were evaluated using one-way or two-way analysis of variance followed by a post hoc Dunnett’s or Sidak’s multiple comparison test.MYPT1 was reported to interact with Rho-kinase 354–775 aa) through its C-terminal region that contains phosphorylation sites by Rho-kinase . We also75 aa thrAs the Rho-kinase-cat-L fragment includes both catalytic domain and some coiled-coil domain , the resWe hypothesized the presence of a Rho-kinase-interacting docking motif within the MYPT1-684–868 aa region. Since 704–715 aa of MYPT1 seemed to be important for the binding to Rho-kinase B, a MYPTWe also examined whether these Rho-kinase-binding MYPT1 fragments inhibit the interaction between Rho-kinase and MYPT1 and resultant MYPT1 phosphorylation. Full-length of GFP-MYPT1 was coexpressed with various MYPT1 fragments in COS7 cells, as shown in The inhibitory effects of MYPT1 fragments on actin stress fiber formation were examined in NIH3T3 cells, to determine whether these fragments actually inhibit Rho-kinase activity in cells. Expression of the MYPT1-684-868 aa or -684-868 aa-4A fragment reduced actin stress fibers, and the effect was milder than that of DN-Rho-kinase C. In conTo examine whether the MYPT1 fragment directly inhibits Rho-kinase activity, an in vitro kinase assay was performed using the S6 rsk substrate peptide as a substrate. The MYPT1-684–868 aa fragment inhibited the Rho-kinase-cat-L activity in a dose-dependent manner A. MYPT1 Since the kinase–substrate interfaces serve as the target of peptide inhibitors such as pseudosubstrate (PS) and docking site/motif, synthesized peptides derived from DM1, DM2, and sequences surrounding phosphorylation sites with alanine substitution  were anaWe further searched for the MYPT1 sequence necessary for the interaction with Rho-kinase, because the PS1 + DM1 peptide failed to suppress Rho-kinase activity, even though the 684–758 aa fragment reduced MYPT1 phosphorylation. We found that a basic amino acid cluster exists (683–691 aa), similar to DM2, at the N-terminus of Ser696/Thr697 C and thaE. coli and subjected to in vitro phosphorylation analyses. Deletion of DM1 or DM3 from the 684–758 aa fragment did not alter the phosphorylation of each fragment by Rho-kinase, whereas deleting both DM1 and DM3 almost completely abolished phosphorylation  close to the phosphorylation site Ser1508 was involved in the Rho-kinase binding , suggestThe MYPT1 of MLCP is one of the physiologically important substrates for Rho-kinase, which broadly regulates cellular contractility . In thisThe C-terminus of the Rho-kinase catalytic domain was thought to be the interface for MYPT1, even though it was not sufficient. Replacing the amino acid of Rho-kinase within the 396–407 aa region by MRCKβ decreased the binding to MYPT1  and kina2–3-X2–6-Φ-X-Φ , with varying distances from the phosphorylation site. The basic and hydrophobic amino acids of the D-motif bind to the acidic patch and hydrophobic groove of MAPKs, respectively. Here, we identified the DM1 (DLQEAEKTIGRS), DM2 (KSQPKSIRERRRPR), and DM3 (RKARSRQAR) sequences of MYPT1, but these sequences are not highly conserved. DM2 and DM3 are rich in basic amino acids, and both are located at the N-terminus of the respective phosphorylation sites  and FXFP-motif, which interact with the different sites of the catalytic domain of MAPKs ,5,24. Thon sites C. Replacon sites , suggeston sites C, implyion sites . The affThe solution structure of human MYPT1-658–714 aa based on the NMR spectroscopy and structural calculation has been reported, in which the MYPT1-658–714 aa consists of three helixes   25]. DM. DM25]. The DMs identified in this study are completely conserved between rat and human MYPT1 (Gene symbol: PPP1R12A). We were intrigued by mutations in these regions and the potential involvement with diseases in human MYPT1. In dbSNP, we found nine, six, and one human missense SNPs in DM1, DM2, and DM3, respectively. Among these missense SNPs, rs1872606231 (E708[709]G), rs1565744239 (I711[712]V), rs1395690034 (G712[713]R), rs1872428296 (S839[841]T), and rs1872426757 (R847[849]G) may alter the affinity of MYPT1 to Rho-kinase (numbers in  indicates amino acid number in rat) . Though In human proteins, sequences of three DMs completely match the corresponding sequences of human MYPT1, as mentioned above, and closely match those of human MYPT2  and MBS85 . Other than the MYPT family members, we cannot find any protein with high similarity to MYPT1 DMs, though some proteins have sequences with partial similarities. Due to the low sequence similarity among the DMs of MYPT1, prediction of the phosphorylation site of substrates by Rho-kinase, especially from the huge shotgun phosphoproteomic data, still seems arduous. We searched for the similar sequences in the already-known Rho-kinase substrates. Basic amino acid clusters, such as DM2 and DM3, are found near the phosphorylation sites of Scrib, MLC, ARHGAP35, MARCKS, and Adducin1. The sequence rich in basic amino acids of Scrib (1451-PLGGGAPVRTAKAERRHQERLRVQSPE-1477) close to the phosphorylation site Ser1508 was confirmed to be involved in the Rho-kinase binding . In addiPseudosubstrate peptides have been utilized as inhibitors for certain kinases, such as PKA, PKC, and CaMKII, which contain consensus sequences with substitution of Ala for phosphorylation sites ,2,6. PepIn summary, we identified novel interfaces (DMs) of MYPT1 with Rho-kinase and found that these DMs contribute to efficient and specific phosphorylation at the proximal sites. Synthetic peptides containing both DM and PS sequences inhibited the activity of Rho-kinase and can serve as potential Rho-kinase-specific inhibitors. Information on docking motifs with consensus sequences will enable us to predict the phosphorylation site of Rho-kinase.