N = 213) conducted the Go/No-Go Task and the Modified Stroop Task. They also completed self-report measures of depression, trait rumination, trait worry, stressors, and aggressive behaviors. Results indicated that response inhibition deficits were positively associated with trait rumination, and this association was mediated by increases in aggressive behaviors and interpersonal stressors. The associations between these variables remained significant even after controlling for depression level. There were no significant direct or indirect associations between attentional inhibition deficits and rumination. These results suggest that response inhibition deficits, among the subcomponents of inhibition, have an indirect positive association with rumination through interpersonal processes. Results also showed nonsignificant differences between rumination and worry in the magnitude of correlation coefficients with the two subcomponents of inhibition. Therefore, it remains unclear whether the positive association with response inhibition is unique to rumination.Previous findings on relationships between inhibition that is a core executive function, and trait rumination have been inconsistent. This inconsistency could be overcome by investigating the association between rumination and the two subcomponents of inhibition: response inhibition and attentional inhibition. This study examined whether and how response inhibition and attentional inhibition were related to rumination as well as worry. University students in Japan ( Rumination is repetitive and passive thinking about one’s depressive symptoms and possible causes and consequences of these symptoms , which is a popular measure of inhibition. Altamirano et al.  indicateo et al.  contradio et al. .These inconsistent results could be explained by a recent, empirically supported model of inhibition. Tiego et al.  proposedHowever, previous studies' findings are limited because they did not compare the relationships of response inhibition and attentional inhibition with trait rumination in the same study and possibly reporting contradictory effects of response inhibition and attentional inhibition on rumination due to differences in sample characteristics. In addition, previous studies did not adequately consider the pathways from each subcomponent of inhibition to rumination. For example, although Hasegawa et al.  showed tThe present study’s primary purposes were to compare the concurrent association of response inhibition and attentional inhibition with trait rumination and to examine possible pathways from these subcomponents of inhibition to rumination in university students. Following Hasegawa et al. , we assuPrevious studies have examined whether depression confounded the relationship between rumination and other factors related to rumination, such as executive functions  participated in this study. We recruited students in this age range to avoid any age effects. All participants were Japanese, with the exception of one participant who was Vietnamese.Beck Depression Inventory-Second Edition  in our sample.Ruminative Responses Scale  and 4 . The RRS is composed of 5 items assessing brooding, 5 items assessing reflection, and 12 depression-related items. Confirmatory factor analysis of the data indicated that the two-factor model comprising of brooding and reflection had an acceptable fit indices range (χ2 (34) = 87.579, p < 0.001; CFI = 0.909; RMSEA = 0.086, 90% CI: ). The fit indices of the two-factor model were insufficient , brooding (α = 0.82), and reflection (α = 0.71) subscales were obtained in our sample.Adequate psychometric properties of the RRS, including good internal consistency and construct validity, as well as moderate test–retest reliability of the total and subscale scores, have been reported  and 5 . Meyer et al. (α = 0.93).r et al.  demonstrr et al.  was usedScale of Life Events in Interpersonal and Achievement Domains  and 4 (often). We calculated scores on the negative interpersonal events subscale and negative achievement events subscale. We obtained adequate internal consistencies for negative interpersonal events (α = 0.82) and negative achievement events (α = 0.79) subscales in our sample.Aggression Scale  and 5 (extremely true of me). This study used the overt aggression subscale score as an index of aggressive behaviors. This subscale is composed of 12 items assessing physical and verbal aggressive behaviors, including, “sometimes I was violent unintentionally,” and “sometimes I was sarcastic and said bad things to others’ faces.” Overt aggression subscale demonstrated good internal consistency (α = 0.84).Go/No-Go Task (GNG): We used the GNG task, which was based on the procedure adopted by Gutiérrez-Cobo et al. , during which time the participants could respond. Then, participants completed the test trial phase that was composed of 4 blocks of 120 trials. The stimulus appeared for 250 ms during each trial in all the blocks. However, the ISIs varied across blocks such that the ISI in a given block was 400, 600, 800, or 1000 ms. The order of the blocks was randomized among participants. Go-trials were 70%, and no-go trials were 30% in each block. The erroneous response rate of each participant in no-go trials (commission error rate) was calculated for each block.r = 0.696, p < 0.001, 95% CI: ).To measure response inhibition, we used the GNG composed of four blocks in which the ISIs differed from block to block and assessed the appropriate ISI of the GNG : The MST based on the procedure adopted by Altamirano et al. .o et al.  was usedStudents interested in this study were individually invited to the authors’ laboratory. First, we obtained their informed consent for participating in the study. Then, participants completed the GNG, the MST, and the CCPT.Z-test was conducted using R. Other analyses were conducted using Mplus 8.3  ≥ 0.95 and root-mean-square error of approximation (RMSEA) ≤ 0.06 as indicators of good fit  test was conducted using SPSS ver. 23 (IBM Corporation), and the χ2 (52) = 77.22, p = 0.013), indicating that missing values were not random. However, it is unlikely that the Full Information Maximum Likelihood (FIML) method’s estimates could be highly biased because there were very few missing data points . In addition, the results of path analyses conducted in this study were approximately similar to those described below, even when the data of the five participants showing Cook’s Ds greater than one in either correlation were excluded. Therefore, we analyzed the data of all the participants.Multivariate outliers were checked using Cook’s As described above, we assumed that response inhibition was indirectly associated with rumination via aggressive behaviors and interpersonal events. We also examined the direct effects of response inhibition on both types of events because response inhibition might be indirectly related to negative interpersonal and achievement events via behaviors other than aggressive behaviors. Moreover, we examined the direct effect of attentional inhibition on rumination following the suggestion by Altamirano et al. . The curSpecific variables of the models might be unsuitable for constructing a latent variable in these analyses. Snyder et al.  indicateZ test for comparing two dependent correlations showed that response inhibition was more strongly correlated with rumination than attentional inhibition . Similarly, Z tests compared correlations of rumination and worry with the two inhibition measures. Results indicated that neither the correlation with response inhibition deficits nor the correlation with attentional inhibition deficits differed significantly between rumination and worry .Descriptive statistics of each measure are shown in Table χ2 (2) = 1.530, p = 0.466; CFI = 1.000; RMSEA = 0.000, 90% CI: ). The residuals of the correlations in this analysis are displayed in Table R2 of this model are illustrated in Fig. We conducted path analysis with FIML to examine whether and how response inhibition and attentional inhibition deficits were related to rumination assessed with the total RRS scores. The model provided an excellent fit to the data .A bias-corrected bootstrap test was conducted to determine the significance of the hypothesized indirect effects. The results indicated significant indirect effects from response inhibition deficits to aggressive behaviors to negative interpersonal events to rumination (χ2 (2) = 1.751, p = 0.417; CFI = 1.000; RMSEA = 0.000, 90% CI: ). The residuals of the correlations in this analysis ranged from − 0.006 to 0.072. Results showed that deficits in response inhibition had a significant positive association with aggressive behaviors  but not with negative interpersonal events  and achievement events . In addition, negative interpersonal events were positively associated with rumination , whereas response inhibition deficits, attentional inhibition deficits, and negative achievement events did not show significant associations with rumination . Each variable explained 3.9% of the variances in aggressive behaviors, 23.6% of the variances in negative interpersonal events, 25.2% of the variances in negative achievement events, and 31.6% of the variances in rumination. Bootstrap tests indicated the 95% CI of the indirect effect from response inhibition deficits to aggressive behaviors to negative interpersonal events to rumination included zero .We also conducted a path analysis with depression as an exogenous variable influencing aggressive behaviors, stressors, and rumination. This model had a good fit for the data (χ2 (2) = 1.434, p = 0.489; CFI = 1.000; RMSEA = 0.000, 90% CI: ). The residuals of the correlations in this analysis ranged from 0.000 to 0.075. Negative interpersonal events showed significant positive associations with brooding , whereas other variables including response inhibition deficits did not show significant associations with brooding . Each variable explained 16.6% of the variances in brooding. Bootstrap tests indicated significant positive indirect effect from response inhibition deficits to aggressive behaviors to negative interpersonal events to brooding .Next, we analyzed the brooding subscale score instead of the total RRS score as the dependent variable. The model provided an excellent fit to the data (χ2 (2) = 1.357, p = 0.508; CFI = 1.000; RMSEA = 0.000, 90% CI: ). The residuals of the correlations in this analysis ranged from 0.000 to 0.075. Results showed that neither variable was significantly related to reflection . Each variable explained 5.4% of the variances in reflection. Bootstrap tests showed the 95% CI of the indirect effect from response inhibition deficits to aggressive behaviors to negative interpersonal events to reflection included zero .We repeated the identical analysis using the reflection subscale score instead of the brooding subscale score. The model provided an excellent fit to the data . Future studies are needed to replicate these findings in different and larger samples. Second, the cross-sectional design of the study precludes identifying causal relationships, which require a longitudinal design. Third, this study used single tasks to measure response inhibition and attentional inhibition rather than latent variables constructed from multiple task performance. Executive function tasks have a task-impurity problem and low test–retest reliability, and therefore, a latent variable approach is ideal for examining relationships between executive functions and other variables of interest, including psychopathological symptoms (Snyder et al.,


Cardiovascular magnetic resonance (CMR) derived fractal analysis of the left ventricle (LV) has been shown in adults to be a useful quantitative measure of trabeculation with high reproducibility and accuracy for the diagnosis of LV non-compaction (LVNC). The aim of this study was to investigate the utility and feasibility of fractal analysis in children.Eighty-four subjects underwent CMR: (1) 28 patients with LVNC  was higher in the LVNC group than in the hyper-trabeculated group: 1.345 (SEM 0.053) vs 1.252 (SEM 0.034), p < 0.001 and higher in hyper-trabeculated group than in controls: 1.252 (SEM 0.034) vs 1.158 (SEM 0.038), p < 0.001. The highest maximum FD was in the apical portion of the LV in the LVNC group,  whereas it was in the mid ventricle in the hyper-trabeculated  and healthy groups . Fractal analysis showed lower intra- and interobserver variability than the Petersen and Jacquier methods.It is technically feasible to perform fractal analysis in children using CMR and that it is quick, accurate and reproducible. Fractal scoring accurately distinguishes between LVNC, hyper-trabeculation and healthy controls as defined by the Petersen criteria. Left ventricular (LV) noncompaction (LVNC) is an important and widely recognized morphological trait consisting of prominent trabeculation, a thin compacted myocardial layer, and deep intertrabecular recesses in some cases –7. The cDiagnosis can be problematic due to the heterogeneity of LVNC and the naturally trabeculated nature of the LV. Diagnostic criteria for both echocardiography and cardiovascular magnetic resonance (CMR) are in current use –13 such bnormal)  and it ibnormal)  and Jacqmentclasspt{minimabnormal) , 17.Fractal analysis is now commonly utilized in other branches of medicine , 19 and It is a semi-automated method, which allows assessment of how an object fills the space. It is used in nature to describe for example the complexity of landscapes  and it hThe aim of this CMR based study was to investigate the utility and feasibility of fractal analysis in the diagnosis of LVNC in the pediatric population.This was a single-centre retrospective study of 84 children < 18 years undergoing a CMR scan between 2011 and 2018: 28 subjects with LVNC, 28 with hyper-trabeculation and 28 healthy controls.All patients with suspected LVNC undergo a CMR scan as part of their initial clinical assessment in our dedicated pediatric cardiomyopathy service. For patients with inconclusive echocardiographic assessment regarding LV hyper-trabeculation and not meeting LVNC echocardiographic criteria, our departmental policy is to assess for possible LVNC using another modality which is preferentially CMR in our practice. The Petersen method is then used to distinguish patients into mutually exclusive groups as follows: patients are included in the LVNC group if the largest noncompacted/compacted (NC/C) layer ratio is measured For the purposes of this study, individuals were included in the control  group, if the CMR scan revealed completely normal findings. The indication for these scans was non-diagnostic echocardiogram, uncertainty about the anatomical structures on echocardiography, syncope or chest pain with low pre-scan probability of being cardiac in origin.The study was approved by the Local Research Ethics Committee. Parents or guardians signed a written consent.2), indexed LV end-systolic volume , LV ejection fraction (LVEF). Papillary muscles and trabeculations were included in the global myocardial mass (and excluded from blood pool) in-line with our published departmental protocol [Contiguous standard short axis cines were performed on a dedicated 1.5 T CMR scanner , with full myocardial coverage and retrospective electrocardiographic (ECG) triggering from breath-held, balanced, steady-state free-precession cines. Typical cine frame time was 40 ms, TE 1.1.ms, field-of-view 320–360 mm frequency encoding (FE) × 240-280 mm phase encoding (PE) directions, acquired spatial resolution 1.7–1.9 mm in FE × 1.7–1.9 mm PE direction with 8 mm slice thickness and 2 mm gap, additionally parallel imaging with 1.7 acceleration factor was used. All images were analyzed with validated software . Volumes and mass measurements were indexed to body surface area (BSA) and the following parameters were calculated: indexed LV end-diastolic volume   with fule-tuning . In caseglobal FDAverage fractal dimension of all short axis slices was recorded as Maximum FD was described as highest FD value obtained from a single slice within the short axis cine stackMaximum FD value from a single slice was also recorded separately within the basal, mid-ventricular and apical slicesFor the purposes of the study, the following parameters were identified:Fractal analysis as above took approximately 3–5 min per patient.Intra- and interobserver variability was assessed by assessing the LV trabeculations using fractal analysis in 30 individuals  using all 3 methods by the same observer  and by different observer (SH for interobserver variability) blinded to the first measurements.https://cran.r-project.org/bin/windows/base/). Descriptive data were expressed as mean  except where otherwise stated. Patient group characteristics were non-normally distributed and compared by the Wilcoxon signed-rank non-parametric test. Correlation between continuous variables was assessed with Pearson’s correlation coefficient. Fractal dimension was compared between the three study populations using Student’s t-test. For the fractal method, area under the receiver operating characteristics (ROC) curve (AUC) was calculated using the method described by Robin et al. [For statistical analysis, a p-value < 0.05 was considered significant. Statistical analysis was performed in R for Windows version 3.5.2  for age, weight, BSA and gender Table . The tabGlobal FD shown in Fig. When maximum FD was compared between all 3 groups at the level of the LV basal, middle and apical thirds separately Fig. .Fig. 3DiThere were different patterns for the maximum FD distribution from base to apex in the LVNC group on the one hand and in the hyper-trabeculated and healthy control groups on the other. In the LVNC group, the maximum FD was in the LV apical third and lowest in the basal third. By contrast, in the hyper-trabeculated and healthy control groups the reverse was true with the highest maximum FD being in the middle part of the LV  0.99, 95% CI 0.98, 1.00). The Petersen method had more intraobserver variability than the fractal method  and the Jacquier method showed the worst intraobserver variability . Bland Altman plots are shown in Fig. Interobserver variability was lower using the fractal method , and considerably higher using the Petersen  and Jacquier methods . Fractal analysis showed very low bias (0.002) and limits of agreement   FD was significantly higher in the Petersen-determined LVNC group than in a hyper-trabeculated group, (2) FD was significantly higher in the hyper-trabeculated group than in healthy controls, and (3) fractal analysis was more reproducible than the Petersen and Jacquier methods.One of the most advantageous features of the FD is its inherent capability to describe the complexity of different objects by single index. In LVNC, FD helps to compute how much of the two-dimensional image space is filled by the contour. It quantifies complexity of geometric patterns. The resulting FD is a unitless quantitative index of how completely the object fills space. FD increases with increased structural complexity. LV FD uses a box-counting method on CMR short-axis cine stacks. As the range of possible FD for a fractal set is dictated by its dimension, the endocardial borders visualized in two-dimensions, are more complicated than simple straight lines, so their FD must be > 1, but because they do not fill two-dimensional space completely, their FD must be < 2. Therefore, the range of possible FD for an endocardial border is between 1 and 2. In LVNC, trabeculations create irregular endocardial border, therefore should generate a higher FD when compared to normal hearts.In contrary to Petersen method, FD is independent of compacted wall thickness which is possibly a confounding factor causing overdiagnosis or underdiagnosis in hypertrophied hearts when there is hypertrabeculation but is more accurate in LVNC with wall thinning  . TherefoRecently fractal analysis has been used in adults to assess the complexity of LV trabeculation in a 2-dimensional setting in order to distinguish LVNC from healthy black and white controls . FractalThe great advantage of fractal analysis, especially in this population, is that it does not require any special sequences and thus does not prolong the scanning time. The whole analysis is performed off-line using the short-axis cine stack, which is always part of the clinical CMR scan and analysis is less user-dependent compared to the Petersen or Jacquier methods.Several groups studied genetics of the LVNC and it is now clear that no specific gene ontology exists. LVNC has been reported in patients with dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, arrhythmogenic right ventricular cardiomyopathy, channelopathies, bicuspid aortic valve, aortopathy, congenital heart defects and in the pediatric population it was also associated with rare genetic syndromes, mitochondrial cardiomyopathy, and Barth syndrome , 31. In As the accuracy of fractal analysis depends on the spatial resolution and image quality of the underlying cine images, it was important to ascertain if the high reproducibility achieved in the adult population  could bThe Petersen method was found to have worse reproducibility than fractal analysis and this might be explained by the fact that this technique although very fast and not requiring additional sequences, is very much reader dependent. Low reproducibility could also be contributing to the fact that Petersen criteria is usually not able to discriminate pathology from physiologic adaptation when the NC/C is borderline . The JacOur results show that the maximum FD should be considered as an alternative and perhaps preferable parameter for establishing the diagnosis of LVNC in children as there was no overlap between the LVNC group and the hyper-trabeculated or normal group. Never less, for patients maximum FD of 1.4 which divides both groups, we propose to use a combined approach of using clinical parameters such as family history, ECG, LV volumes, LVEF, genetics data, Petersen criteria and LGE. Global FD calculated as the mean of FDs in every slice of the LV is less able to group the patients correctly which may have been caused by the fact that the basal slices have less trabeculation even in the noncompacted group. A similar distribution pattern was also found in the adult population . WhereasIn the future it will be important to further ascertain the clinical utility of fractal scoring by in particular evaluating if fractal scoring is strongly associated with outcome and biomarkers. In this context, recent work by Wang et al. reported that increased apical fractal dimension (≥ 1.325) was an independent predictor of all-cause mortality and aborted sudden cardiac death in patients with hypertrophic cardiomyopathy after adjustment for the European Society of Cardiology predictors and LGE .In this study we utilized the Petersen method as the current “gold standard” for CMR diagnosis of LVNC. We acknowledge that the many drawbacks to this method make it imperfect—in fact this was the very rationale for this study. Ultimately, we do believe an alternate automated method with low intraobserver and interobserver variation such as fractal scoring would be preferable. However, in presenting for the first time an alternative we felt the current standard should be utilised to ascertain the groupings.We have shown that fractal analysis is a promising and reproducible imaging method for the diagnosis of LVNC in the pediatric population. Maximum FD in particular may serve as a good marker for distinguishing between children with LVNC and normals and those with hyper-trabeculation (but not noncompaction). This may serve as a useful adjunct in the diagnosis of LVNC in children, either as a sole method or complementary to the Petersen method. The clinical utility now needs to be assessed in a long-term outcome study acknowledging that there may be a benign group of LVNC. Further research in this area is warranted.


However, the mechanism of NO● interference with 15LOX/PEBP1 activity remained unclear. Here, we use a biochemical model of recombinant 15LOX-2 complexed with PEBP1, LC-MS redox lipidomics, and structure-based modeling and simulations to uncover the mechanism through which NO● suppresses ETE-PE oxidation. Our study reveals that O2 and NO● use the same entry pores and channels connecting to 15LOX-2 catalytic site, resulting in a competition for the catalytic site. We identified residues that direct O2 and NO● to the catalytic site, as well as those stabilizing the esterified ETE-PE phospholipid tail. The functional significance of these residues is supported by in silico saturation mutagenesis. We detected nitrosylated PE species in a biochemical system consisting of 15LOX-2/PEBP1 and NO● donor and in RAW264.7 M2 macrophages treated with ferroptosis-inducer RSL3 in the presence of NO●, in further support of the ability of NO● to diffuse to, and react at, the 15LOX-2 catalytic site. The results provide first insights into the molecular mechanism of repression of the ferroptotic Hp-ETE-PE production by NO●.We recently discovered an anti-ferroptotic mechanism inherent to M1 macrophages whereby high levels of NO Recent years have brought attention to ferroptosis, an iron-dependent form of regulated cell death implicated in a broad range of diseases ,2, and tcis-5,8,11,14-ETE or shortly ETE, is the preferred substrate of these non-heme iron-containing enzymes; but under inflammatory conditions, such as those stimulated by interleukin-13/4, the formation of the complex 15LOX/PEBP1 shifts the substrate-specificity of 15LOX from free AA to AA esterified into PE (or ETE-PE). The AA-esterification of PE mostly occurs at the sn-2 acyl chain leaving the sn-1 chain for saturated or mono-unsaturated acyls, predominantly palmitic (C16:0), stearic (S) (C18:0), and oleic (C18:1). Of interest below will be 1-SA-2-ETE-PE, i.e., PE with stearic acid (SA) and arachidonic acid (AA or ETE) at the respective sn-1 and sn-2 chains, which will be shortly called SAPE. 15LOX-catalyzed peroxidation of SAPE at its sn-2 chain results in the production of 15-HpHETE-PE [The role of 15LOX in the onset of ferroptosis is now well-established ,4,5,6. U2+-independent phospholipase A2β (iPLA2β) [● [●’s reactivity towards different radical intermediates of lipid peroxidation has been discovered more than three decades ago [● inhibits lipid peroxidation by lipoxygenase and cyclooxygenase [As a multistage process, ferroptosis is regulated by several enzymatic events occurring independently or concertedly: (i) biosynthesis of PUFA-PE peroxidation substrates by acyl Co-A synthetase 4 (ACSL4) and lysophosphatidylcholine acyltransferase (LPCAT) ; (ii) pr(iPLA2β) , which cLA2β) [● . Notablyades ago . Furtherxygenase .● indeed suppresses the production of HpETE-PE by 15LOX-2/PEBP1, while promoting the formation of nitrosylated ETE-PE. As the 15LOX-2/PEBP1 complex, rather than 15LOX-2 alone, catalyzes the oxidation of 15-ETE-PE (rather than free ETE), we focused here on the interactions of NO● with SAPE and15LOX-2/PEBP1, and on its competition with the lipoxygenase substrate O2. Notably, the precise delivery of molecular oxygen to form a peroxyl radical from the carbon-centered radical of ETE at the 15LOX-2 catalytic site occurs via a specialized channel, whose structure and accessibility is altered upon binding of ETE to the enzyme [● peroxidation of SAPE by 15LOX-2/PEBP1 and the structural dynamics of the oxygen delivery process to the catalytic Fe3+ in the 15LOX-2/PEBP1 complex and the competition with NO●, in the presence and absence of the ETE-PE substrate compared to free ETE in 15LOX-2. Our study provides a mechanistic description of the time-evolved interactions between 15LOX-2 (bound to PEBP1) and its ligands, the ETE-PE- and PEBP1-induced changes in 15LOX-2 conformation or catalytic site accessibility which alter the delivery of O2 and NO●, and the competition between O2 and NO● for binding the active site. Several key residues mediating the events prior to peroxidation and the interference of NO● to repress them are identified, the critical roles of which are consolidated by in silico saturation mutagenesis experiments and comparisons with recent work.In the present study, we first performed a lipidomics analysis to observe that NOe enzyme . With th● on the production of the pro-ferroptotic signal, 15-HpETE-PE, by 15LOX-2/PEBP1, we incubated one of the lipoxygenase isoforms, 15LOX-2, and PEBP1 in the presence of an NO●-donor, propylamine (PAPA) NONOate [● at a constant rate with a decay half-time of 15 min. We found that NO● strongly suppressed the production of hydroperoxide of 1-SA-2-ETE-PE (HpETE-PE) (● (To examine the effects of NO NONOate . This dopETE-PE) a. DetailE-PE)  direct reaction of NO● with the ETE-PE carbon-centered intermediate, and (ii) potential presence or occupancy of the O2-binding site and channeling path by NO● or a competition between the two small molecules—O2 and NO●—interfering with the peroxidation of the oxidizable substrates. To explore the latter mechanism, we analyzed the changes in the conformational state and dynamics of 15LOX-2/PEBP1 invoked upon binding of the oxidizable substrate, as well as O2 and NO●.Combined, these results suggest that at least two mechanisms contribute to the observed suppression of ETE-PE peroxidation by NO●. First, we explored how the accessibility of the catalytic site of 15LOX-2 to O2 and NO● molecules is altered upon complexation with PEBP1. Next, we investigated how the presence of NO● affects O2-binding and channeling to the catalytic pocket. We further analyzed the effect of oxidizable substrates ) on the accessibility of the 15LOX-2 catalytic site to O2 and NO●, in the presence and absence of PEBP1. Overall, we conducted 10 sets of molecular dynamics (MD) simulations in duplicate (or triplicate) runs of 150 nanoseconds (ns) under different conditions, summing up to total simulation duration of 3.6 microseconds in explicit water, as summarized in the We performed structure-based modeling and simulations to investigate the molecular basis of the experimentally observed reduced activity of 15LOX-2/PEBP1 in the presence of NOE1–E3), which could potentially allow for the entry of the radicals, O2 or NO●, to potentially access the catalytic cavity of the enzyme  and, therefore, inaccessible to O2 and NO●. In contrast, E1 served as the main access point to a tunnel leading to the catalytic site, for both O2 and NO● , H553 (on helix α27), and the C-terminal isoleucine, I676 . Simulate enzyme a: E1, neentrance ; E2, at nterface a; and E3and A599 d. Althoutic site d. SuppleE1 was lined by Y154, N155, G157, W158, I421, I435, F438, and S439 , the small molecules moved deeper towards two attractive sites, binding sites S1 and S2, that arrested them for extended durations  with NO● or O2 for sufficiently long durations , S430 and V603 near the surface  the cluster I216, I604, F561, C564, and A565 that initially bound O2/NO● for ~40 ns; and (ii) the cluster L607, L610, L420, V426, and V427 that retained the molecules for ~11 ns. The third site that serves as a bridge between E3 and catalytic site partially overlaps with S2: it contains the residues L246, E364, F365, H368, E369, and L570 that bound O2/NO● for ~40 ns. Thus, S2 serves as an attractor for O2 or NO● binding entering through either E1 or E3. Two highly conserved residues therein, F365 and E369, will be shown below to play an important role in binding or redirecting the O2 and NO● molecules.The entrance  surface d. These E1 and E3), binding to S1 and S2, or insertion towards the catalytic site (mainly hydrophobic clusters), is given in A summary of all these residues involved in various roles, entry , until the departure of O2 from the catalytic site, consistent with the experimentally observed ability of NO● to inhibit the peroxidation of SAPE.Simulations with the substrate revealed an interesting effect which arises upon the complexation 15LOX-2 with PEBP1, namely a competition between Otic site , Movie 1● and O2 molecules, starting from first encounters with O2 , followed by the arrival of NO● to the catalytic site and the competition between NO● and O2 for binding the catalytic pocket residues  and yellow (right)), finally ending with the dislocation of O2, while NO● remained stabilized near C15 and Fe3+ ion. Among residues ligating (successively or competitively) both O2 and NO● during this trajectory, we note the highly conserved residues P365 and E369 in site S2, and the hydrophobic cluster V603, A606, L609, and L610, revealing that O2 and NO● channeled to the catalytic site through Entrance 3 in this case.The panel j in 2/NO● molecules (see Methods), among those located within 20 Å of the Fe3+ ion. 2 and NO●, and the corresponding number of runs in which such contacts are observed. Clearly 15LOX-2 residues at Entrances 1 and 3 (E1 and E3) are detected among them, along with sites S1 and S2 residues, as indicated by the bars between panels b and c. Of particular importance is the long helix α12-14 (F365-L379) comprising both S1  and S2  residues also noted [2 and NO● in the absence of PEBP1 but become inaccessible upon PEBP1 binding.We further carried out a statistical analysis of four independent runs to identify the 15LOX-2 residues which made frequent and long-lived contacts with Oso noted  in the aso noted . The higso noted  also tak2 and NO● co-localize within the catalytic site  chain of SAPE and to the 15LOX-2 residues L172-A179. At a higher NO● concentration, however, this effect was partially suppressed and a few NO● molecules took a place near C15 atom of SAPE in the catalytic site  competitive binding of NO● to the catalytic site in the presence of PEBP1  PEBP1 increases the affinity of 15LOX-2 to bind both Oof PEBP1 a is expetic site .2 molecule to be positioned in proximity (<7.5 Å) of the carbon C15 in the arachidonoyl chain of SAPE, while the C13 in the arachidonoyl chain of SAPE would approach (by <7.5 Å) the iron ion at the catalytic site. We analyzed the MD trajectories to examine whether such poses were sampled. We identified a total of 121 and 538 poses, respectively, for PEBP1-bound and -unbound 15LOX-2/SAPE (S1) and A606 (at E3), were distinguished by their high tendency (75% of MD snapshots) to coordinate SAPE, in both systems. Furthermore, α2-helix residues N181, Y185, and G189 exhibited a high propensity to coordinate the substrate, Y185 playing a major role in the presence of PEBP1; and N181 in the absence. Additionally, PEBP1 D144 and P186 contributed to stabilizing the SAPE.Lipid peroxidation by 15LOX-2 requires OX-2/SAPE a that sa2/NO● taking part in E1 or E3, or associated sites S1, S2, and other clusters (of mostly hydrophobic residues) paving the way to the catalytic site  to 1 (most damaging or pathogenic). The predictions are based on the evaluation of the sequence-, structure- and dynamics-properties of 15LOX family proteins in comparison to the features observed in more than 20,000 missense variants used as learning dataset [The present study points to several residues implicated in initial entry, channeling, and binding of Otic site . As a teRhapsody . This ma dataset ,24.The results for residue segments of interest are presented as a heatmap in 2/NO●) or substrate (SAPE) are predicted to be critical to function. Our analysis in fact revealed that the residues labeled along the upper abscissa, written in boldface in E3, W158-P159 and I421 at E1, and N413, R417, L421, and L374 at site S1. Among them we note that some play a dual role of coordinating the substrate SAPE too  are found to be tolerant to mutations  the residues involved in mediating the interactions with the ligand  are particularly sensitive to substitutions. Examination of the structure shows that this region, composed of a long helix  spans the entire structure at the center, making contacts with both c pocket . Its sca●. We had previously shown that RAW 264.7 M2 macrophages are susceptible to ferroptosis when their phospholipid hydroperoxide-specific glutathione peroxidase is inhibited by RSL3 [● [● in the close proximity of carbon-centered radicals formed by lipoxygenase.Encouraged by these results, we next examined whether the nitrosylated PE products are formed in cells in which ferroptosis is inhibited by NO by RSL3 . In thes RSL3 [● . Both Fe● potency to directly control ferroptosis in macrophages and microglia, and distantly in several neighboring  cells [● in ferroptosis further increased by the possibility of using NO● donors for attenuating, suppressing, or delaying ferroptotic death [● with lipid radical intermediates , have been considered [●’s inhibitory effect remained elusive.Among different aspects of ferroptosis, its physiological regulation by mechanisms other than the well-established effect of GPX4, attracted particular attention in recent years . Among tl) cells . Interesic death . Althougnsidered , the exa●’s potential regulation of this catalytic complex has emerged as an important consideration. Our experiments in a model biochemical system with purified recombinant proteins directly demonstrate the ability of NO● to inhibit the catalysis of ETE-PE oxidation to 15HpETE-PE by the complex but not by 15LOX-2 alone. This suggests that structural features specific to the 15LOX-2/PEBP1 complex could account for this inhibitory effect. Given that O2 is delivered to the 15LOX-2 catalytic site via a channel regulated by the binding of the oxidizable substrate, the role of structural changes in the 15LOX-2/PEBP1/SAPE in controlling O2 delivery and possible competition with NO● became and even more fascinating yet unsolved puzzle. The present LC-MS data showing nitrosylation of ETE-PE, i.e., direct interaction of NO● with a carbon-centered radical, rather than with the peroxyl radical intermediate, LO2●, provide further evidence for the direct interference of NO●.Given the reported participation of 15LOX-2/PEBP1 complex in the generation of 15HpETE-PE that serve as a pro-apoptotic agent, NO●-SAPE) than 15LOX-2 alone suggesting that a larger number of NO● molecules were able to enter the catalytic pocket of PEBP1-bound 15LOX-2 compared to free 15LOX-2. In accord with these observations, our simulations showed the higher propensity of NO● molecules to access the catalytic site in the presence of PEBP1 bound to 15LOX-2. In contrast, in the absence of PEBP1 and at small concentration of NO●, NO● were attracted to the sn-1 tail of SAPE or grouped in a new cavity formed after loop L172-A179 reorganization in 15LOX-2 alone simulations. The experimentally observed formation of nitrosylated-ETE-PE catalyzed  by 15LOX-2 alone [● at the catalytic site. The in silico studies at similar concentrations of both molecules, could not detect any NO● at the catalytic site. However, an increase in the number of NO● molecules in the MD system lead to a few NO● accessing the active site, explaining the nitrosylated-ETE-PE formation in 15LOX-2 alone. We also note that the turnover rate for 15LOX-2 is rather slow (~8.5–25/s). Thus, the current simulations mainly provide insights into the mechanistic aspects of NO● actions, rather than a quantitative description of the overall kinetics.The lipidomics experiments showed that the 15LOX-2/PEBP1 complex produced greater amount of the nitrosylated product  was not altered significantly by NO●, in line with the lower accessibility to the 15LOX-2 catalytic site (in the absence of PEBP1) shown in MD simulations.Our simulations unambiguously showed that this effect, occurring in the presence of PEBP1 is indeed due to the ability of NO2/NO● molecules with 15LOX-2 residues during their journey towards the catalytic site. There are two available entrances to the catalytic cavity. The first (E1) is near Y154-W158 loop the flexibility of which is presumably limited by a proline, P159, highly resistant to substitutions. The second (E2) is near S430, S573, P595, A599, and L603. Both entrances provide access to the catalytic site through intra-protein channels that mediate the translocation of the small molecules. Our analysis highlighted the importance of L374, N413, L420, I421, and L610 in enabling efficient communication with the catalytic site. Detailed analysis of the substrate interaction site with 15LOX-2 and PEBP1 at the most favorable geometric positioning for lipids peroxidation also revealed crucial interactions with selected residues  that formed a substrate-binding epitope.Finally, our work provides a detailed mechanistic description of the interactions of OS1 and in the cluster of hydrophobic residues that connect the entrance E3 to the catalytic site. Likewise, the mutation N426M in 5LOX, which is the homologous counterpart of the site S2 T431 in 15LOX-2, gives rise to a loss of activity when associated with F360W and A425I [E3) reduced catalytic activity and altered the stereo-selectivity of the oxygenation reaction [Comparison of the critical residues with prior observations made for lipoxygenase family variants lends support to the functional significance of several sites identified here. For example, loss of 15LOX-2 activity has been observed in the variant A416D , a residnd A425I . Likewisreaction . It is a●, the enzymatic machinery is still in place, but it cannot effectively produce HpETE-PE. Even though the direct reaction of NO● with the ETE-PE carbon-centered intermediate takes place, albeit at a very low level, our analysis strongly suggests that the observed suppression of ETE-PE peroxidation by NO● in the 15LOX-2/PEBP1 complex is mostly due to the occupancy of the O2-binding site or channeling path by NO●, and a competition between the two radicals (O2 and NO●) thus resulting in the reduced, if at low physiological NO● amounts not completely abrogated, ETE-PE peroxidation. Interestingly, our highly sensitive redox lipidomics analysis did not reveal the formation of nitroxygenated ETE-PE derivatives. This suggests that the direct chemical reaction of NO● with O2—very effective in gas and liquid phases [● to repress the peroxidation of free and esterified PUFAs by 15LOX-2/PEBP1 could be exploited in designing anti-ferroptotic therapies.The overall analysis identified not only key sites, but their intricate couplings to enable the catalytic activity of 15LOX-2 complexed with PEBP1. In the presence of NOd phases —is stron● and O2 molecules were performed for each structure (15LOX-2 and its complex with PEBP1) using the NAMD [●, O2, and covalently bonded Fe3+ were created based on bound O2 and heme group using Gaussian [Conventional full-atomic MD simulations ,48,49 wethe NAMD  softwarethe NAMD  force fithe NAMD ) at physthe NAMD . The binthe NAMD  ligand-pthe NAMD . CHARMM Gaussian  package.Gaussian  for visuGaussian ,59,60 foGaussian  3.0 withThe Rhapsody tool  was usedg for 15 min at 4 °C. Then, 20 μL of the supernatant was transferred to an auto sampler vial, and 5 μL was injected into the LC-MS/MS system. LC-MS/MS analysis was performed as described previously.Human recombinant 15LOX-2 and PEBP1 were recombinantly expressed an purified as described previously ,63. 15LO2 in DMEM or RPMI (ATCC) supplemented with 10% heat-inactivated fetal calf serum  and 50 U ml−1 penicillin–streptomycin . RAW 264.7 macrophages were polarized by to M2 stage by incubating in DMEM containing 10% FBS, 50 U mL−1 penicillin–streptomycin, and IL-4 (20 ng mL−1) for 48 h. For ferroptotis experiments, cells were incubated with RSL3 (0.5 µM), 2 µM), +Fer-1 (~400 nM), or ±DPTA NONOate (25 µM), for 5 h. Cell death was determined by flow cytometry.RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC). Cultured at 37 °C and 5% CO−1 on a Dionex Ultimate 3000 HPLC system . The column was maintained at 35 °C. Analysis was performed using gradient solvents (A and B) containing 10 mM ammonium acetate. Solvent A contained propanol:hexane:water  and solvent B contained propanol:hexane:water . All solvents were LC–MS grade. The column was eluted for 0–23 min with a linear gradient of 10–32% B; 23–32 min using a linear gradient of 32–65% B; 32–35 min with a linear gradient of 65–100% B; 35–62 min held at 100% B; 62–64 min with a linear gradient from 100% to 10% B followed by and equilibration from 64 to 80 min at 10% B. The instrument was operated with the electrospray ionization probe in negative polarity mode. Data was analyzed using Quan Browser of xcalibur software (Thermo Fisher).MS analysis of phospholipids was performed on an Orbitrap mass spectrometer (Thermo Fisher). Phospholipids were separated on a normal phase column (Luna 3 µm Silica (2) 100 Å, 150 × 2.0 mm, Phenomenex, Torrance, CA, USA) at a flow rate of 0.2 mL min


Protein kinases play a significant role in cellular activation procedures by exhibiting a vivid selection in the target, as well as recognizing and phosphorylating them. Extracellular signal-regulated kinase 2 (ERK2) is one of the main kinases in the mitogen-activated protein kinase (MAPK) signaling cascade and engages in dynamically regulating the activities of signaling proteins and physiological processes, including cell proliferation, differentiation, adhesion, migration, and survival. Predicting collective dynamic and structural motions in biological macromolecules is pivotal to obtain a better understanding of the majority of biological processes. Here, through molecular dynamic simulation and normal mode analysis, we investigated ERK2 conformations, in the forms of active (phosphorylated), inactive (unphosphorylated), and in a complex with its substrate, ribosomal protein S6 kinase alpha-1 (RSK1), to determine functional characteristics. Our finding demonstrated that ERK2 plays a switch role in the regulation of pathways. In the case that this protein kinase is in the active form, all critical regions shift to be prepared to accept the substrate and catalytic action. Meanwhile, inactive ERK2 shows contrasting results in which all motions tend to close the catalytic site and cease the phosphorylation action in the MAPK cascade. These findings are in line with those from other similar studies and provide us with novel molecular target regions and recent details on how this mechanism works. DFG, αC-helix orientation regarding the active site position and distance between K54 and Glu71 in the active and inactive states of ERK2. The RAS/RAF/MEK/ERK cascade is an important MAPK signaling pathway and includes a small G protein (RAS) and three protein kinases . RAS protein could be phosphorylated through the binding of various growth factors to the cell surface receptors .3,4 Activated RAS recruits and activates RAF, a serine/threonine-protein kinase. Phosphorylated-RAF then activates MEK1/2 (MAPK/ERK kinase), dual-specificity protein kinase, and activated MEK1/2 phosphorylates ERK1/2. Finally, activated-ERK1/2 affects several substrates and different transcription factors, which would alter the gene expression, whether in cytosol or nucleus.5,6Mitogen-activated protein kinase (MAPK) pathways regulate diverse functions in cells. These pathways are activated by various stimuli, continue into the nucleus, and phosphorylate numerous proteins affecting gene expression, metabolism, division, and survival of the cell. Dysregulation of MAPK cascades leads to cancer and other human diseases.7 Human ERK1 and ERK2 consist of 379 and 360 residues, respectively, and like other kinases have a glycine-rich loop, catalytic loop, activation loop, P + 1 loop, D-domain, and F-domain. In the development of mice, the erk1 gene is dispensable; nonetheless, the ablation of the erk2 gene results in embryonic death. Due to the higher expression levels of ERK2, it has been studied more than ERK1.8,9 There are 160 target molecules downstream of ERK1/2, such as different transcription factors , cytosolic phospholipase A2, ternary complex factor (TCF) family, cytoskeletal proteins, apoptotic proteins, and ribosomal S6 kinase (RSK) family. RSK, a protein-serine/threonine kinase with influential roles in cell regulation, is one of the cytosolic substrates of ERK1/2. Growth factors, polypeptide hormones, neurotransmitters, and chemokines induce the activation of the RSK pathway.10 The only available crystal structure for ERK1/2 in the complex with its substrates is ERK2–RSK1  with PDB ID 4NIF11,12 , which causes additional specificity for these proteins.2 Fig. S1. The RSKin silico methods, including molecular dynamics simulation and normal mode analysis have been performed on three forms of ERK2 including active-ERK2, inactive-ERK2, and ERK2 in a complex with RSK1 in order to deeply understand and explain the functional details behind the structure and dynamics of ERK2. It is noteworthy that the motions of each one of these three forms were precisely examined and compared with each other. To prove the results, normal mode analysis was performed by calculating the lowest-frequency modes with a high degree of collectivity.In this study, 2.2.12ERK), inactive (3SA0), and ERK2–RSK complex (4NIF) forms for molecular dynamics (MD) simulations. Structural investigations were performed by Swiss-PDB viewer 4.10 and Chimera1.12.18,19 VMD 1.9.1 has also been used to visualize ERK2's structural dynamics during MD simulations.20 All the structures have been depicted in ribbon and surface representations in order to provide a clear and simplified image of the proteins without considering all-atom details.Crystal structures of ERK2 were obtained from Protein Data Bank in active , and then the coordination and topology files were saved for the next steps of MD simulations.Molecular dynamics simulation was performed using AMBER 14 with ff14SB force field.23Minimization of ERK2 with ions and water was performed in two steps. First, water and ion and then the whole system were minimized with 4000 steps . Non-bonded interactions were calculated at a cutoff distance of 10 Å by PME  method in periodic boundary conditions.24 Then, the equilibration was done for 600 ps in the NPT ensemble. Finally, production MDs were performed in 300 ns for all forms with the NPT ensemble. Additionally, the SHAKE algorithm was used for hydrogen atoms. The coordinates were then saved every 20 ps. All of the simulations were repeated three times, each for 300 ns with random initial velocities.The system was heated from 0 to 300 K for 150 ps, using Langevin thermostat in NPT ensemble.2.3cpptraj from AMBER Tools 15 was used.22 The root mean square deviation (RMSD), root mean square fluctuation (RMSF), and the radius of gyration (Radgyr) were calculated regarding the initial structure (eqn (S1)–(S3)In order to analyze the MD production, 2.4ElNemo) server.25 Lowest-frequency modes (modes 7–12) with a high degree of collectivity were calculated. Distance fluctuation maps of residues and mean square displacement were depicted as a measure of structural flexibility.The normal mode analysis of ERK2 was performed by Elastic Network Model (3.3.12ERK), inactive (PDB ID 3SA0), and complex states with RSK1 (PDB ID 4NIF), have been simulated. RMSD values of different forms of ERK2 during simulation are illustrated in 2+ (blue graph), was more stable in comparison with other structures. An increase in the RMSD value in inactive-ERK2, without ATP–Mg2+ (red graph), was vividly visible at the beginning of the simulation, which is due to the conformational changes of the structure. The RMSD of ERK2–RSK1 (performed in 300 ns) is also shown in ERK1/2 are activated by upstream proteins such as RAF, RAS, and MEK. Active-ERK2, also, affects downstream proteins. RSK1 is one of the ERK2's substrates that is activated after the ERK2–RSK1 association. This activated RSK1 is transferred to the nucleus and phosphorylates transcription factors. Based on dynamic changes in ERK2 protein kinase, the active (PDB ID 32–37 αC-helix (61–74), catalytic loop (147–154), activation segment ), and KIM (a 31-residue sequence insertion within the kinase domain (248–277)), which are functionally important, represented various levels of flexibility. The active forms of ERK2 (blue and green) and the inactive forms (pink and red) had a different flexibility pattern during the simulations. In order to activate the MAPK pathway, ERK2 interacts and phosphorylates RSK1, such that Thr573 from RSK1 was phosphorylated by Asp149 in the catalytic loop of ERK2 and these residues had a low RMSF value  had the lowest Radgyr during the simulation  revealed the tightest packing owing to the movement of the C-lobe and N-lobe in a closer orientation in the structure. On the other hand, inactive forms of ERK2 (red and pink) had lower compactness. In the ERK2–RSK1 complex, the value of Radgyr for RSK1 fluctuates between 21.5 to 22.5 Å, meanwhile, Radgyr for ERK2 is more stable and has a small variation around 21.5 Å.According to the n Fig. S2. The act187YVATRWYR194) is located in the middle and ends with an APE motif .7 All MAP kinases only phosphorylate substrates with proline in the P + 1 site.26–28The activation segment has important roles in both substrate binding and the catalytic activity of protein kinases. The activation segment in most of the kinases begins with a DFG motif (Asp167–Phe168–Gly169 in ERK2) and activation lip . The P + 1 loop  were the components of the K/D/D motif. 2+ and coordination of ATP  and Asp167 in DFG motif (binds to Mgn of ATP  and S5†.2+ and coordinates the charges of phosphates in ATP), whereas in the inactive state it moves away from the active site (“DFG-aspartate-out”).29 These fluctuations are also observed in active and inactive ERK2. As illustrated in The DFG motif in the activation segment of ERK2 has different conformations in active and inactive forms. In the active-ERK2, the aspartate side chain of DFG (Asp167) moves towards the active site called the “DFG-aspartate-in” (binds to Mg7 As shown in Another important and conserved region is αC-helix (residues 61–74), which is located in the N-lobe of ERK2. The αC-helix in active-ERK2 faces into the active site, known as “αC-helix-in”, while its orientation is altered to “αC-helix-out” in inactive-ERK2. It is noteworthy that ERK2 is active in both “αC-helix-in” and “DFG-in”. Besides, Glu71 in the αC-helix is connected to Lys54 (in β3 N-lobe), which is considered a salt-bridge. In active-ERK2, the salt-bridge was established and the connecting residues were getting closer to each other during the simulation, meanwhile, in inactive-ERK2, these two residues were getting far from each other. In other words, the kinase was inactive in the absence of the salt-bridge.In order to evaluate the integrity of the ERK2–RSK1 complex, the distances between Lys54–Glu71 that are involved in the salt bridge in ERK2, as well as Asp149 in ERK2 and Thr573 in RSK1, have been calculated (Asp149 is involved in the interaction with Thr573 to prepare RSK1 for phosphorylation). As represented in Fig. S6,Every substrate possessed one or two docking domains for interaction with a specific kinase. The D-site and F-site are two docking domains that help ERK1/2 to interact with up/down-stream proteins. DRS, D-recruitment site  and FRS, F-recruitment site,  in ERK2 were related to the D-site and F-site docking domains, respectively. The accessible surface areas of DRS and FRS are shown during the simulation in 3.2Distance fluctuation maps (DFM) were calculated to investigate variations of the Cα distances in the active, inactive, and complex forms of ERK2. According to DFM plots for modes 9 and 11, the distance between C-lobe and N-lobe was decreased in the active form, while this distance was increased in the inactive form. According to the DFM of mode 7 for complex form , the dis4.30–32In the present study, we applied molecular dynamic simulation and normal mode analysis to evaluate the conformations of ERK2 in the forms of active (phosphorylated), inactive (unphosphorylated) and in the complex with RSK1 to determine its functional characteristics. MD simulation and normal mode analysis have been used as tools to investigate the local flexibility and large-scale conformational transitions. The results of the study show that ERK2 switches between open and closed forms during the simulation. In the active state, all critical regions of ERK2 were rearranged to be prepared for accepting the substrate and catalytic action. In contrast, all motions in the inactive-ERK2 lead to closure of the catalytic site. These findings were consistent with previous studies.2+ (red graph) had a higher RMSD value and showed an unstable form compared to other structures. Whereas, active-ppERK2 (blue graph) in the absence of ATP  in order to occupy the active site via covalently bound phosphates.28 Structural investigation of the activation segment demonstrated that in the active-ppERK2, this segment tends to move inside and to close to the N-lobe; while, in the inactive form, it tends to be close to the C-lobe. In other words, the active-ppERK2 has an open active site to recognize its substrates.28 Similarly, our study suggested that in the active-ppERK2 this segment moved towards the cavity of the enzyme and slightly approached the N-lobe. The activation loop was open and allowed the P + 1 loop to find the interaction site, whereas, in the inactive-ERK2, the P + 1 loop folded and decreased the accessibility of Thr187. This conformation blocked the phosphorylation and activation of ERK2's substrates because it impedes the P + 1 loop to find the interaction site  that act as the catalytic core of protein kinases.38 Lys54 cooperates with Glu71 to produce a salt-bridge and Asp149, in a catalytic loop, traps the proton from the OH group from substrates and facilitates the nucleophilic attack of oxygen on the γ-phosphate of ATP.39 The second aspartate, Asp169, in the DFG motif, binds to Mg2+ ion to coordinate negative charges of phosphates groups from ATP.7 Given our findings, it was observed that the orientation of these residues changed during the simulation. Therefore, the distance between Lys54 and Glu71 declined and the salt-bridge was established , CD , and hydrophobic groove  in DRS moved closer to each other during simulation; whereas, in the inactive-ERK2 these regions were far from each other. Furthermore, FRS is only accessible in the active-ERK2 that probably prepares ERK2 for the interaction with RSK1, while this region was covered by the activation loop in the inactive form  in ERK2 along with Thr573 (activation loop) in RSK1.from NMA  suggeste5.via DRS in ERK2 and D-docking domain in RSK1, the catalytic reaction could be implemented. As ATP-binding site was taken up by ATP–Mg2+ in ppERK2, the protein kinase was catalytically active, and Asp149 located in the active site of ERK2 began to phosphorylate the Thr573 in the activation loop of RSK1.The MAPK signaling pathway includes several key protein kinases, which play a major role in cellular processes regulating cell growth, proliferation, differentiation, apoptosis, and migration. MAP kinases are specifically controlled in these activities such as their interactions with substrates and activating enzymes. In this study, we employed computational methods to investigate the structural and functional features of ERK2 in different forms such as active, inactive, and also in the complex with its downstream partner called RSK1. This survey showed how computational approaches could be practical for examining the biological behavior of ERK2 and helped us to find out the dynamics of this protein in its different forms. The results, with a focus on the dynamical properties of ERK2, confirmed the same behavior for ERK2 with other protein kinases. The salt-bridge, conformation of αC-helix and DFG, as well as open activation loop, are some features for distinguishing active and inactive kinases. According to the phosphorylation sites in the activation lip of ERK2, the catalytically active and inactive forms of the protein can be defined. When Thr185 and Tyr187 were phosphorylated by MEK1/2, known as the upstream protein of ERK2, the activation loop was open so that N- and C-lobes get close to each other. As a result, the salt-bridge was established between K54 and E71 in the N-lobe as these regions moved towards each other. In addition, the distances between K/D/D decreased, αC-helix and DFG faced into the active site (αC-helix-in and DFG-aspartate-in), and eventually, the entire structural conformations were ready to interact with downstream substrates including RSK1. Following the interaction of ERK2 with RSK1, 28,44In conclusion, the activation lip, as the critical regulator of the ERK2 activity, underwent conformational changes during the simulation. In the inactive structure, the activation lip was folded to block the P + 1 site and forced the neighboring structures, namely, KIM, the C-terminal extension, and helix C, into an open conformation. On the other hand, in a phosphorylated state, the lip was rearranged and exerted domain closure, resulting in the formation of the proline-directed P + 1 site. Moreover, the other changes occurred in L16, which caused close conformation as well as remodeling of phosphorylation-dependent interactions with other macromolecules. These changes in L16 also led to the formation of the ppERK2 dimer, which is physiologically important. In addition, phosphorylation of Tyr and Thr in the lip directly affected the lip's conformation in active and inactive ERK2. According to the previous studies and our findings, it is assumed that a specific conformation of activation lip in different ERK2's states contributes to the regulation of MAP kinase pathway and this region is conserved for activating point mutations in the enzyme.45,46The ultimate goal of this study was to understand the function and dynamical behavior of ERK2 in complex with RSK1 in order to design inhibitory molecules to disrupt their interaction for blocking MAPK signaling pathway as to control cancer cell growth and proliferation as a potent and novel therapeutic method which has already been proposed in other studies for other cases.Bcl-2B-cell lymphoma 2c-MycProto-oncogene MycCREBcAMP response element-binding proteinDFMDistance fluctuation mapsDRSD-recruitment siteeIF4BEukaryotic translation initiation factor 4BElNemoElastic network modelERK2Extracellular signal-regulated kinase 2Ets1ETS proto-oncogene 1FRSF-recruitment siteJNKc-Jun N-terminal kinaseK/D/DLys/Asp/AspKIMKinase insert domainMAPKMitogen-activated protein kinaseMDMolecular dynamicsNF-κBNuclear factor kappa-light-chain-enhancer of activated B cellsPDBProtein data bankgyrRadRadius of gyrationRMSDRoot mean square deviationRMSFRoot mean square fluctuationRSK1Ribosomal protein S6 kinase alpha-1RTKReceptor tyrosine kinaseTCFTernary complex factorTIF1ATranscription initiation factor 1AThere are no conflicts to declare.RA-011-D1RA01020D-s001


Tarsometatarsal (TMT) arthrodesis is a common operative procedure for end-stage arthritis of the TMT joints. To date, there is no consensus on the best fixation technique for TMT arthrodesis and which joints should be included.Thirty fresh-frozen feet were divided into one group (15 feet) in which TMT joints I-III were fused with a lag screw and locking plate and a second group (15 feet) in which TMT joints I-III were fused with 2 crossing lag screws. The arthrodesis was performed stepwise with evaluation of mobility between the metatarsal and cuneiform bones after every application or removal of a lag screw or locking plate.P < .05). Additional application of a locking plate caused further stability in every TMT joint (P < .05). An additional crossed lag screw did not significantly increase rigidity of the TMT II and III joints (P > .05). An IM screw did not influence the stability of the fused TMT joints. For TMT III arthrodesis, lag-screw and locking plate constructs were superior to crossed lag-screw fixation (P < .05). TMT I fusion does not support stability after TMT II and III arthrodesis.Isolated lag-screw arthrodesis of the TMT I-III joints led to significantly increased stability in every joint (Each fixation technique provided sufficient stabilization of the TMT joints. Use of a lag screw plus locking plate might be superior to crossed screw fixation. An additional TMT I and/or III arthrodesis did not increase stability of an isolated TMT II arthrodesis.We report the first biomechanical evaluation of TMT I-III arthrodesis. Our results may help surgeons to choose among osteosynthesis techniques and which joints to include in performing arthrodesis of TMT I-III joints. Only 2 recent clinical studies focused on the fixation technique for TMT I-III arthrodesis.5 Buda et al concluded that isolated plate fixation significantly increased the nonunion rate after midfoot arthrodesis compared to crossed screw fixation. Ettinger et al found a lower nonunion rate using locking plate plus screw fixation. Two previous biomechanical studies examined midfoot fusion for neuroarthropathic feet and for ligamentous Lisfranc joint injuries.12 However, neither of these studies focused on recent fixation techniques for arthritis of the TMT joints. There is no biomechanical study that compares current common fixation techniques using crossed screws or locking plates with lag screws. Further, it remains unclear to what extent the number of fused TMT joints stabilize the adjacent, nonfused TMT joints.The tarsometatarsal (TMT) joint line can be divided into 3 columns, including a medial (first TMT joint), central (second and third TMT joint), and lateral (fourth and fifth TMT joint) column. For primary or posttraumatic TMT arthritis, TMT arthrodesis of the medial and central column (I-III) is a standard operative procedure.The present study compares crossed lag screws with locking plate and lag screw for TMT I-III fusion. The aim of this study was to determine a superior fixation construct with respect to stability for TMT I-III arthrodesis. In addition, stability in relation to the number of fused TMT joints was analyzed.Thirty fresh-frozen cadaver feet  were used for this biomechanical study. None of the feet showed any relevant deformities or signs of previous surgery. The mean age of the donors was 75.1 ± 11.4 years. Twelve left and 18 right feet were prepared. Twelve donors were female. All feet were randomly assigned to 2 groups (groups 1 and 2) with 15 feet each. There were no demographic differences between the 2 groups. The specimens were placed in a custom-made frame with the heel on the ground in neutral position, enabling the application of 300 N axial loading and 10 N traction to each respective tendon in neutral position of the ankle joint.All feet were thawed at room temperature before experiments. The tibiae were embedded in a 2-component polyurethane casting resin at the proximal ends . The Achilles, posterior tibial, and peroneal tendons were prepared proximal to the ankle joint and fixed with 1 clamp each. The biomechanical setup and testing were performed according to a previously published protocol, which evaluated naviculocuneiform fixation techniques.For rotational evaluation of the metatarsal (MT) and cuneiform (CN) bones against each other, the NDI Optotrak Certus System  was used. This system performs an optical measurement with active markers with a resolution of 0.01 mm. Three reference markers were attached to the frame forming a coordinate system fixed in space. Individual marker clusters consisting of 3 markers each were inserted in the MTI-III and the CN I-III via Kirschner wires. The total angle of rotation was measured from the 3 rotational components as the length of the rotation vector between 2 bones .9Biomechanical testing of each group was performed in the same manner. Every operative step was first evaluated without any tendon pull or axial loading (mode 1). Afterward, the same operative step was evaluated with an application of 300-N axial loading on the shank and 10-N tendon pull force each for the posterior tibial tendon, the peroneal tendons, and the Achilles tendon (mode 2). The amount of rotation between mode 1 and 2 for each joint (TMT I-III and the intercuneiform I/II and II/III joints) was the primary outcome parameter. A decreasing amount of rotation was defined as an increased stability of the arthrodesis. To increase reliability of the results, modes 1 and 2 were performed in every group at each operative step to prevent perturbation of the inserted markers during implant application or removal. All procedures were performed by an experienced foot and ankle surgeon.Arthrodesis was performed using Aptus 2.8 TriLock plates and CCS 5.0 cannulated double-threaded lag screws . Prior to stepwise TMT arthrodesis, the first measurement was performed in the native state to define a baseline for every cadaver foot. The stepwise TMT arthrodeses were performed in the following manner: With the markers inserted, the TMT I-III joints were prepared. In group 1, first a CCS 5.0 double-threaded lag screw and second an Aptus 2.8 TriLock plate were inserted separately in each TMT joint I-III, beginning with the first TMT joint . In grouThen, all implants were removed step by step, beginning with the IM screw and TMT I arthrodesis to evaluate residual stability of the TMT II and III arthrodeses and second, the TMT III arthrodesis to evaluate residual stability of the isolated TMT II arthrodesis. After removal of the implants in TMT III arthrodesis, each foot was measured again in the natural state without any arthrodesis to provide a comparison to the initial natural conditions. Every step was recorded radiographically to ensure correct implant positioning  and 3. TU test was used to compare the 2 fixation techniques. Statistical significance was defined as a P value <.05.Data collection and analysis was conducted in R . Values are expressed as median and range. The statistical analysis was performed using a Wilcoxon signed-rank test for paired data on an interval scale, that is comparison of the different conditions. A Mann–Whitney P < .001). A TMT I arthrodesis did not lead to additional rigidity of the second and third TMT joint.The application of a locking plate and lag screw for TMT I arthrodesis led to significantly increased stability of the first TMT joint (P < .001). With an additional locking plate, further significant increase in stability was detected (P = .007). Removal of TMT I and III arthrodeses did not decrease the rigidity of the TMT II arthrodesis.Applying a single lag screw for TMT II arthrodesis significantly increased the stability of the second TMT joint (P = .003). With an additional locking plate, stability was further significantly increased (P < .001). Removal of the TMT I arthrodesis did not decrease the rigidity of the TMT III arthrodesis.The application of a single lag screw for TMT III arthrodesis significantly increased the stability of the third TMT joint (P > .05).A I-II IM screw did not lead to additional stability in any of the TMT joints, nor in the intercuneiform or intermetatarsal joints (P < .05).Removal of each implant led to increased mobility in the respective joints, comparable to the initial values (P < .001). A TMT I arthrodesis did not lead to additional rigidity of the second and third TMT joints.The application of 2 crossed lag screws for TMT I arthrodesis led to significantly increased stability of the first TMT joint (P = .0067). With an additional crossed lag screw, there was an increase in stability that was not statistically significant (P = .064). Removal of TMT I and III arthrodeses did not decrease the rigidity of the TMT II arthrodesis.Applying a single lag screw for TMT II arthrodesis led to significantly increased stability of the second TMT joint (P = .064). With an additional crossed screw, there was a small increase in stability but it was not statistically significant (P = .1688). Compared with the initial values of the TMT III joint, an arthrodesis of 2 crossed screws significantly increased stability (P < .001). Removal of the TMT I arthrodesis did not decrease the rigidity of the TMT III arthrodesis.The application of a single lag screw for TMT III arthrodesis led to increased stability of the third TMT joint that was not statistically significant (P > .05).A I-II IM screw did not lead to additional stability in any of the TMT joints, nor the intercuneiform or intermetatarsal joints (P < .05).Removal of each implant led to increased mobility in the respective joints, comparable to the initial values . For TMT III arthrodesis, lag-screw and locking plate constructs showed significantly higher stability compared with crossed lag-screw fixation (P = .0007).For the first and second TMT arthrodeses, there was no significant difference in stability between the 2 fixation techniques , whereas Ettinger et al reported a lower nonunion rate after locking plate plus compression-screw fixation compared to 2 crossed screws fixation. Two previously performed biomechanical studies focused on fixation of the first and second TMT joints.12 Marks et al reported higher loads-to-failure of a plantar plate and lag-screw construct compared to isolated screw constructs. In our study, TMT joints II and III were initially fixed with an isolated lag screw in both groups. Application of an additional locking plate led to significantly increased stability in the second and third TMT joints in group 1, whereas an additional lag screw did not significantly decrease movement in the respective TMT joints in group 2. This might indicate higher rigidity of locking plate and lag-screw constructs. However, we could detect the superiority of locking plate and lag-screw constructs only for the third TMT joint (P < .05).Two recent clinical studies focused on midfoot arthrodesis. Buda et al compared TMT I arthrodesis constructs with or without an additional I-II IM screw and reported a greater radiologically improved IM angle and hallux valgus angle in patients with an additional I-II IM screw. In their cohort, the TMT I arthrodesis was performed with a locking plate and cross screw from MT I into intermediate cuneiform constructs instead of a screw fixation of MT I and II. Our testing setup did not focus on the IM and hallux valgus angles, which might have improved after the I-II IM screw as well. Further, a I-II intercuneiform screw or cross screw might have decreased movement in the respective joints in our testing. Biomechanical and clinical studies should be performed to analyze the effects of an intercuneiform screw.In our experiments, an additional I-II IM screw did not lead to further stability in any of the analyzed joints. One recent study by Langan et al stated in their clinical study that the number of treated columns did not affect the nonunion rate, which supports our findings. However, in their cohort, they did not distinguish between the type of TMT arthrodesis performed . Further comparative clinical studies are needed to support our findings.Some patients have isolated TMT II, TMT III, or combined TMT II and III arthritis. To our knowledge, there is no previous study analyzing TMT stability in relation to the number of fused TMT joints. In our analysis, stability of the TMT II arthrodesis remained constant, independent of whether an additional TMT I or III arthrodesis existed. Further, an additional TMT I arthrodesis did not influence the stability of a TMT III arthrodesis. Thus, isolated TMT II or TMT II and III arthrodeses seem to have no adverse effects on stability. These findings are of high clinical relevance and indicate that isolated TMT II or III fusion may not increase risk of nonunion. So, in cases of an unaffected TMT I joint, the TMT I can be left intact and does not have to be fused for stability. Appropriately, Buda et al Our procedures were designed to ensure stepwise measurements of the performed arthrodesis. Thus, we did not do load-to-failure testing. A randomization of the fixation sequence to counter possible ordering effects was not deemed feasible, because it could result in a test order in which a single screw would have to be removed and installed multiple times, which would have drastically affected its stability and subsequently introduced additional bias. An additional testing with the heel off could have shown greater impact on the TMT movements, as this means the greatest loads on the TMT joint line. Third, the utilized setup captured only rotational motion and not translational motion. However, because of the loading of the specimen and the type of fixation, predominantly rotational motion can be expected. At last, we performed axial loading with only 300 N, which is not comparable to physiological loads in vivo. In pilot studies, a higher axial load (800 N) led to wearing out of the cadaver feet throughout the testing protocol. Further, no differences were detected in the TMT joint rotation ratios comparing axial loading with 800 and 300 N. With a reduced load and additive tendon pull, the final measurements differed only minimally from the baseline measurement, which confirmed that our specimens were not worn out.Our study has some limitations that have to be addressed. First, this was a cadaver study. Differences in bone quality might have biased our results. Compared with other biomechanical studies, we did not perform maximum-load-to-failure testing. Other than analysis and comparison of 2 different fixation techniques, our study design evaluated effects on stability dependent on the number of fused TMT joints, according to a previously published protocol.Isolated lag-screw fixation was significantly inferior to 2 lag screws or lag-screw and locking plate constructs. An additional TMT I and/or III arthrodesis did not increase the stability of an isolated TMT II arthrodesis.Click here for additional data file.Supplemental material, sj-pdf-1-fai-10.1177_10711007211033541 for Biomechanical Evaluation of Tarsometatarsal Fusion Comparing Crossing Lag Screws and Lag Screw With Locking Plate by Sarah Ettinger, Lisa-Christin Hemmersbach, Michael Schwarze, Christina Stukenborg-Colsman, Daiwei Yao, Christian Plaass and Leif Claassen in Foot & Ankle International


Inhibitory neurons innervating the perisomatic region of cortical excitatory principal cells are known to control the emergence of several physiological and pathological synchronous events, including epileptic interictal spikes. In humans, little is known about their role in synchrony generation, although their changes in epilepsy have been thoroughly investigated. This paper demonstraits how parvalbumin (PV)- and type 1 cannabinoid receptor (CB1R)-positive perisomatic interneurons innervate pyramidal cell bodies, and their role in synchronous population events spontaneously emerging in the human epileptic and non-epileptic neocortex, in vitro. Quantitative electron microscopy showed that the overall, PV+ and CB1R+ somatic inhibitory inputs remained unchanged in focal cortical epilepsy. On the contrary, the size of PV-stained synapses increased, and their number decreased in epileptic samples, in synchrony generating regions. Pharmacology demonstrated—in conjunction with the electron microscopy—that although both perisomatic cell types participate, PV+ cells have stronger influence on the generation of population activity in epileptic samples. The somatic inhibitory input of neocortical pyramidal cells remained almost intact in epilepsy, but the larger and consequently more efficient somatic synapses might account for a higher synchrony in this neuron population. This, together with epileptic hyperexcitability, might make a cortical region predisposed to generate or participate in hypersynchronous events. Epilepsies are thought to be associated with neuronal hypersynchrony, resulting in the generation and maintenance of paroxysmal activity, such as interictal spikes and seizures. Understanding the role of different neuron types in the generation of synchronies is crucial to identify what makes a brain region predisposed to generate epileptic events. A finely tuned balance of excitatory and inhibitory neuronal activity is required for the emergence of different physiological oscillations and synchronous events ,2, constSynchronous population activity (SPA) emerges spontaneously in the surgically removed brain tissue of both epileptic and non-epileptic (tumor) patients, in vitro ,12,13,14Inhibitory interneurons innervating the perisomatic region of principal cells are known to efficiently control the output, while inhibitory cells terminating on the dendritic region of principal cells regulate their input . Being aSimilar to rodents , PV is pCB1R is present both in the monkey and human neocortex, with a different laminar distribution across different brain regions . CB1R-stAcetylcholine receptors are widely distributed in the brain, and the activation of both ionotropic nicotinic (nAChR) and metabotropic muscarinic receptors (mAChR) is implicated in numerous cognitive functions, such as arousal, learning, and memory. The activation of the nAChRs enhances GABAergic inhibition arriving to both excitatory and inhibitory cells in the hippocampus and the neocortex  through The aim of this study was to describe the alterations in the distribution and the output connections of the two types of perisomatic inhibitory cells in the human neocortex in epilepsy, as well as to elucidate the possible role of these neuron types in the generation of synchronous population activity. We examined the distribution of PV- and CB1R-positive elements in the human epileptic and non-epileptic neocortex and made a quantitative electron microscopic analysis concerning the perisomatic inhibitory input of supragranular pyramidal cell with a focus on PV- and CB1R-positive axon terminals. Furthermore, we investigated the participation of the two perisomatic inhibitory cell types in the emergence of SPA by selectively modulating the cholinergic system.2+-binding protein present in one group of perisomatic inhibitory cells in the human neocortex  µV to 18.10 [12.20–31.21] µV  was added to the bath solution. Before all drug application a baseline was recorded , and all drug effects were compared to this baseline. Frequency and LFPg values of the baseline were considered to be 100%, when determining drug effects. All pharmacological agents were washed out . We have to note that mAChR activation also increases pyramidal cell excitability and firing ,76, and with CCh . Regardip > 0.05), it became similar to values when CCh was applied alone: 0.82 [0.72–1.19] Hz , AF-DX 116 could largely prevent the effects of CCh: the recurrence frequency changed to 0.90 [0.87–1.07] Hz, (83 [71–96] %), which is comparable with the baseline , though it was still significantly lower than during the baseline  in NoEpi and to 63.87 [53.55–76.51] µV (87 [83–91] %) in ResEpi tissue, although these changes were found to be significant only for ResEpi slices  in NoEpi and 4.93 [4.21–5.31] µV (111 [86–135] %) in ResEpi slices  AM-251 could not induce significant changes to restore the effects of CCh: the recurrence frequency was 0.83 [0.02–0.94] Hz . When analysing LFPg amplitude of individual SPA events, we found it to be significantly higher during the co-application of AM251 and CCh than during CCh alone, in both patient groups (p < 0.0001 both in NoEpi and ResEpi cases). However, both values were still significantly lower than during the baseline  and ResEpi tissue , compared to physiological solution. These values were not significantly different from those when only CCh was applied . The application of AM-251 alone did not cause any change in the recurrence frequency and in the LFPg or MUA amplitudes of the SPAs compared to the baseline . The apThe preservation of the overall perisomatic inhibitory input and the ratio of PV+ inhibitory input of neocortical pyramidal cells in epilepsy contrasts to the hippocampal dentate gyrus of temporal lobe epileptic (TLE) patients, where the perisomatic inhibitory input of dentate granule cells has notably increased together with the enhancement of the CB1R+ inhibitory boutons  and withAltogether, more profound changes occur in the epileptic hippocampus considering perisomatic inhibition, than what we have observed in the epileptic neocortex . Other rModulation of the muscarinic receptors modifies the behaviour of excitatory cells, not only that of perisomatic inhibitory interneurons. In the healthy brain, the activation of M1 type mAChRs transiently inhibits neocortical pyramidal cells , which iThe knowledge about the initiation mechanism of neuronal synchronies is crucial to understand the possible differences between physiological and pathological synchronies. Perisomatic interneurons—as the most potent cells to inhibit large neuron populations—are indeed efficient in shaping synchronous activities such as sharp-wave ripples ,90 and iIn the present study, we examined this hypothesis with two different approaches: with pharmacological modulation and quantitative electron microscopic analysis of perisomatic inhibition of neocortical pyramidal cells. Our results showed that perisomatic inhibitory cells indeed contribute to the generation of SPA. By comparing the LFPg amplitudes of the SPA events, we found that modulating the activity of both perisomatic interneuron types partly restored the effect of CCh, both in NoEpi and ResEpi samples (LFPg amplitudes in ACSF > in CCh + antagonist > in CCh). However, when comparing the averages, preventing the effect of CCh on PV+ cells could significantly elevate LFPg amplitude only in ResEpi but not in NoEpi tissue. This latter finding tightly fits to the results of the electron microscopic analysis: only in ResEpi samples, in regions generating SPA, axons of the PV+ (but not the CB1R+ or non-stained) basket cells have been significantly changed. The size of somatic PV+ boutons has significantly increased, together with the decrease in the number of PV+ synapses arriving to the pyramidal cell bodies. The increase in the active zone size is associated to a higher neurotransmitter release, and thus, to a more powerful synaptic transmission . The morOur pharmacological and electron microscopic results suggest that perisomatic inhibitory cells contribute to the generation of SPAs but do not have an exclusive leading role. This supports our previous studies, where we showed that both excitatory and inhibitory cells are needed for the generation and they both participate in the initiation of SPAs ,16. The The patients were operated in the National Institute of Mental Hygiene, Neurology and Neurosurgery . We received written consent from all patients. Our protocol was approved by the Hungarian Ministry of Health and by the Regional and Institutional Committee of Science and Research Ethics of Scientific Council of Health (ETT TUKEB 20680-4/2012/EKU) and performed in accordance with the Declaration of Helsinki.n = 1 patient) and temporal (n = 11 patients) lobes. All patients suffered from pharmacoresistant epilepsy  for 22 ± 15 years on average. Epileptic patients were diagnosed with several different pathologies, such as focal cortical dysplasia, cortical gliosis, tumors of glial origin, hippocampal sclerosis, and stroke induced lesion. In the cases of four patients, the obtained tissue was in the seizure onset zone, whereas in the remaining eight patients the seizures invaded that region, but the seizure focus was elsewhere , temporal (n = 5 patients), parietal (n = 3 patients), and occipital (n = 1 patient) lobes. Thirteen patients were diagnosed with tumors of glial (n = 8) or other origins , equilibrated with 5% CO2 in 95% O2 . Neocortical slices of 500 µm thickness were cut with a Leica VT1000S vibratome . They were then transferred and maintained at 35–37 °C in an interface chamber, perfused with a standard physiological solution containing (in mM) 124 NaCl, 26 NaHCO3, 3.5 KCl, 1 MgCl2, 1 CaCl2, and 10 D-glucose, equilibrated with 5% CO2 in 95% O2 . If not mentioned separately, Molar Chemicals Ltd.  was the manufacturer of the chemicals.Tissue was transported from the operating room to the laboratory (located in the same building) in a cold, oxygenated solution containing (in mM) 248 D-sucrose, 26 NaHCOThe extracellular local field potential gradient (LFPg) recording was obtained as described previously . BrieflyThe presence and the exact location of the SPA was noted in case of every slice. If a stable SPA was observed (persisting without evident changes for more than 10 min), pharmacological experiment was performed. The non-selective acetyl choline receptor (AChR) agonist carbachol (CCh) was applied first  to decrease the GABA release from the perisomatic inhibitory cells . After aData were analyzed with the Neuroscan Edit4.5 program , and home written routines for MATLAB . The microelectrode covered all layers of the neocortex. Usually, channels 1–12 were in the supragranular, channels 13–15 in the granular and channels 16-23 were in the infragranular layers. Channel positions were determined according to the thickness of the neocortex of the given patient and corrected if necessary.Detection of the SPA was performed on LFPg records after a Hamming window spatial smoothing and a band-pass filtering between 1 and 30 Hz . Note that SPA is the totality of ‘SPA events’ occurring in a given recording (on average 286 ± 214 SPA events/recording). SPA events were visible usually on 5 to 16 channels, and SPA events larger than two times the standard deviation of the basal activity were detected and included in the analysis. The largest amplitude LFPg peak of the events was chosen as time zero for averaging. The location and the recurrence frequency of the SPA events were determined in each file during the whole recording session. To examine significance in the LFPg amplitude change induced by the different pharmacological agents, we used both the averaged LFPg amplitude and the LFPg amplitude of all SPA events occurring in a given recording. The latter will be called ‘individual LFPg amplitude’. The channel showing the largest LFPg amplitude on the averaged LFPg amplitude was chosen for the analysis of the individual LFPg amplitudes. Multiple unit activity (MUA) was calculated from the LFPg using standard techniques . LFPg anData are presented as median (first to third quartiles). Changes compared to physiological solution  were given in percentage of the values in physiological solution (median (first to third quartiles)).2O2 in TBS for 10 min. TBS was used for all the washes (3 × 10 min between each serum) and for the dilution of the antisera. Non-specific immunostaining was blocked by 2% normal goat serum and 2% normal horse serum for one hour. The primary antibodies were applied for two days at 4 °C. For visualization of the immunopositive elements biotinylated anti-rabbit (in case of CB1R) or anti-mouse  IgG  was applied as secondary antibody (2 h), followed by avidin-biotinylated horseradish peroxidase complex . The immunoperoxidase reaction was developed by 3,3′-diaminobenzidine tetrahydrochloride dissolved in Tris buffer (pH: 7.6), as a chromogen. Sections were osmicated , dehydrated in ethanol and mounted in Durcupan .Immunostainings were performed to visualize the two types of perisomatic inhibitory cells: the PV- and the CB1R-positive interneurons, as well as to verify the laminar structure and the possible tumor infiltration or signs of dysgenesis in the neocortex . NeocortSince CB1R has a different laminar distribution across different cortical areas , for theThe number of the examined cell bodies varied between 7 and 18 among the samples, with an average of 11.7 ± 3.7 per region of interest. Interneuronal and glial somata were excluded from the analysis, based on their morphological properties . Inhibitt-test. The synaptic coverages (µm synaptic active zone/100 µm soma perimeter) and the numbers of synapses/100 µm soma perimeter values failed the normality test, therefore statistical differences were assessed with Mann–Whitney U test or Kruskal–Wallis ANOVA for comparing two or multiple groups, respectively. In cases of significant differences obtained with Kruskal–Wallis ANOVA, the p values were corrected with the Bonferroni adjustment. In order to test for unequal proportions in contingency tables we used Chi-square test.We determined the statistical significances with the aid of the program Statistica 13 . The lengths of the synaptic active zones  followed normal distribution (verified with the Kolmogorov–Smirnov and Lilliefors test), and statistical significances were examined with the Student’s t-tests with a Bonferroni adjustment. Welch’s t-test was done to reveal presumable differences  between the NoEpi and the ResEpi group. The significance level was set to p = 0.05.The statistical significances were examined with the aid of the R software . None of the data sets followed normal distribution (verified with the Shapiro–Wilk test). One-way repeated measures ANOVA was done to reveal presumable differences between the effects of the applied pharmacological agents  within each patient group (NoEpi or ResEpi). Pairwise differences were examined by post-hoc multiple pairwise paired