Hundreds of millions of general anesthesia are performed each year on patients all over the world. Among these patients, 0.1–0.2% are victims of Accidental Awareness during General Anesthesia (AAGA), i.e., an unexpected awakening during a surgical procedure under general anesthesia. Although anesthesiologists try to closely monitor patients using various techniques to prevent this terrifying phenomenon, there is currently no efficient solution to accurately detect its occurrence. We propose the conception of an innovative passive brain-computer interface (BCI) based on an intention of movement to prevent AAGA. Indeed, patients typically try to move to alert the medical staff during an AAGA, only to discover that they are unable to. First, we examine the challenges of such a BCI, i.e., the lack of a trigger to facilitate when to look for an intention to move, as well as the necessity for a high classification accuracy. Then, we present a solution that incorporates Median Nerve Stimulation (MNS). We investigate the specific modulations that MNS causes in the motor cortex and confirm that they can be altered by an intention of movement. Finally, we perform experiments on 16 healthy participants to assess whether an MI-based BCI using MNS is able to generate high classification accuracies. Our results show that MNS may provide a foundation for an innovative BCI that would allow the detection of AAGA. Waking up during a surgery is a haunting experience, both for patients, who consider it as the worst in their lives  , the literature clearly shows that their classification rate is lower than that of a synchronous BCI with triggers  and show that it is a very promising approach. Indeed, previous studies have shown that a painless stimulation of the median nerve induces an ERD during the stimulation while an ERS appears after the stimulation  verify that median nerve stimulation generates desynchronizations (ERD) and synchronizations (ERS); (ii) confirm that they are modulated by an intention of movement; and (iii) demonstrate that a classification based on this phenomenon would be more effective than conventional classification based on modulations generated by an intention of movement vs. resting state. In order to achieve the above, we recorded 128 EEG signals from 16 voluntary healthy subjects who had performed 3 motor tasks  and reacted to a MNS. To show the influence of a MI on the ERD and ERS generated by a MNS, we computed time-frequency and topographic maps and a classification based on MNS+MI and MNS only. Our results indicate that a MI significantly modulates the ERDs and ERSs generated by a MNS and also that classification based on MNS is more efficient than conventional classification based on MI vs. rest. These results are promising for creating a BCI that detects AAGA.Sixteen right-handed healthy volunteers  were recruited for this study. All voluntary subjects satisfied the inclusion criteria . This experiment followed the statements of the WMA declaration of Helsinki on ethical principles for medical research involving human subjects . In addition, participants signed an informed consent which was approved by the ethical committee of Inria  as it satisfied the ethical rules and principles of the institute.The aim of this research is to investigate the occurrence of motor patterns under 4 different conditions : real movement (RM), motor imagery (MI), median nerve stimulation during a motor imagery (MI + MNS), and median nerve stimulation during rest (MNS) . The firThe RM condition (C1) consisted of an isometric grasp between the thumb and the index finger on a pointer button . A low fFor the MI condition (C2), subjects had to imagine the previous movement, i.e., they had to try to feel a maximum of sensations caused by the real movement, but without any movement. Similarly to condition 1, a low frequency beep indicated when the subject had to start the motor imagery, the grasping MI was maintained during 2 s, then a second beep indicated the end of the imagined task .During the MI + MNS condition (C3), subjects had to perform a motor imagination while their median nerve was stimulated 750 ms after the start of the motor imaginary task . UniformThe MNS only condition (C4) consisted of a series of stimulation of the median nerve during rest. We placed the two electrodes of stimulation on the wrist according to the standards  installation of the EEG cap (40 min); (2) selection of the intensity of median nerve stimulation needed to produce a micro movement between the thumb and index finger (10 min); (3) execution of RM, MI, MI + MNS, and MNS in runs during which participants had to perform the different motor tasks (60 min); (4) uninstallation and debriefing (10 min).The study contained 4 conditions: real movement (RM), motor imagery (MI), median nerve stimulation during motor imagery (MI + MNS), and stimulation only (MNS). These conditions were completed on the same day and divided into two runs of 26 trials each, representing 52 trials for each condition. The duration of one trial was 8 ± 1 s . The runFor the entirety of the experiment, the subject were seated in a comfy chair including a leg rest, with their right arm resting on a pillow, a presentation remote placed in their right hand. The subject didn't actively hold the remote, it was simply resting in their hand with the button placed under their thumb. Subjects were asked to keep their eyes closed .OpenViBE platform  active electrode and Driven Right Leg (DRL) passive electrode located over the parietal lobe. Impedance was kept below 10 kΩ for all electrodes to ensure that the background noise in the acquired signal was low. An external electromyogram (EMG) electrode was added in order to verify that there was no movement during the MI task.EEG signals were acquired using the All offline analyses were performed using the EEGLAB toolbox  from the EEGLAB toolbox was used to validate differences in terms of time-frequency of this ERSPs.To analyze the differences between all four conditions, we performed an event-related spectral perturbation (ERSP) analysis between 8 and 35 Hz using EEGLAB. We used a 256 point sliding fast Fourier transform (FFT) window with a padratio of 4 and computed the mean ERSP 2s before the task to 7 s after the task. ERSP allows to visualize event-related changes in the average power spectrum relative to a baseline of 1.5 s taken 2 s before the auditory cue for C1 and C2, and 2 s before stimulation for C3 and C4 . A surrogate permutation test  from the EEGLAB toolbox was used to validate differences in terms of localization of this ERSPs. In addition to this analysis, we applied a false discovery rate (FDR) correction test in order to clarify how the false discovery rate was controlled for multiple comparisons. This test consists of repetitively shuffling values between conditions and recomputing the measure of interest using the shuffled data. It was performed by drawing data samples without replacement and is considered suitable to show the difference between MI + MNS and MNS conditions  for MI + MNS and MNS conditions  since it is considered one of the most effective and accurate techniques used to extract events , and the rest time windows started 3 s before the go signal. For MI + MNS and MNS conditions, we selected a time window of 3 s starting 0.5 s before the median nerve stimulation for all trials of both conditions. The recorded EEG signals were bandpassed using a 5th-order Butterworth filter between 8 and 30 Hz. For each classes, we collected a total of 52 trials.t-test (two-sided) to show the significant difference about accuracy obtained for MI vs. Rest and MI + MNS vs. Rest with the TS + LR classifier .We computed the performance of four different classification methods in a 4-fold cross-validation scheme. The first one uses a Linear Discriminant Analysis classifier (LDA) trained and evaluated using Common Spatial Pattern (CSP) features generated from the first and last 4 CSP filters , synchronization/control of the median nerve stimulator and sound beep generation was designed with OpenViBE software  and the subsequent motor task (pressing or releasing the button). It also includes the number of trial rejected because of acquisition artifacts.For the Real Movement condition, the reaction time between the first beep and the movement start was 0.5948 s ± 0.1929. The reaction time between the second beep and the movement stop was 0.5038 s ± 0.1174. These two reaction times can be considered as normal in the light of the literature on this domain . Due to the presence of artifacts acquired during the experiment, we used an artifact rejection script to remove the most important ones. We removed 125 (15%) trials for the RM condition, 119 trials  for the MI condition, 114 trials  for the MNS condition, 138 trials  for the MI+MNS condition. The removed artifacts are homogeneously distributed among the subjects.The time-frequency maps display the signal's power evolution and are useful to establish the frequency and time windows in which ERSP appears .For the Real Movement condition (C1), we can observe two separate ERDs during the motor task in both mu (7–13Hz) and beta (15–30Hz) band . In the p < 0.05) shows a significant difference between the MI constant ERD and the RM interrupting ERS. Additionally after the motor task in C1, there is an ERS in the mu band which doesn't exist for C2. Finally, PMBR for MI seems weaker than the rebound for RM.Throughout the Motor Imagination condition (C2), a continuous ERD occurs in both mu and beta band . It starp < 0.05) but the ERD (250–500 ms after the MNS) is very similar. The MNS-generated beta rebound appears less powerful than the one from C3 and, instead of a return to baseline, a continuous mu ERD last until the end of the motor task. Finally a third rebound appears in both frequency bands 1,200 ms after the motor task.In the case of median nerve stimulation (MNS) during rest state (C3), a powerful and robust ERS appears immediately (0–250 ms) after the stimulation in low mu (7–10 Hz) and low beta (15–22 Hz) . For theAnalysis of these time-frequencies maps showed that both mu (7–13 Hz) and beta (15–30 Hz) bands were impacted in term of synchronization/desynchronization in all four conditions. Since the previous results for C1 and C2 are consistent with the literature and the purpose of this study is to discriminate C3 and C4, we will only look into the last two conditions. Consequently, a larger frequency band (8–30Hz) was chosen to analyse the ERD and ERS localization. According to this In accordance with the results obtained from the time-frequency and topographic analyses, the ERD and ERS modulations have been computed for three frequency bands, mu: 8–12 Hz, beta: 15–30 Hz and mu+beta band: 8–30 Hz for all subjects. The In the mu frequency band, a desynchronization appears and reaches –15% during both MI task . This observation confirms that the ERD created by the MI task isn't impacted by the following MNS (besides the slight PSR in C4) in the mu band. Logically, this desynchronization doesn't exist for the MNS condition. After the motor task, a slight rebound appears for MNS and MI + MNS condition.The ERD in the beta frequency band behaves similarly to the ERD in the mu band, only C2 and C4 display this desynchronization. However, during MI + MNS, the ERD is shorter  than the one in MI only. Logically, during C3 no ERD appears. As seen on the topography and time-frequency figures, an ERS appears for all conditions 3 and 4 after the stimulation. This ERS is partially diminished for MI + MNS but is followed by a stronger post-motor task rebound , also present for C2.If we merge the two frequency bands, the behavior of ERD and ERS is particularly interesting since the difference between condition 3 and 4 is strong on a 0–3,000 ms time window. On the same note, after 3,000 ms, the condition 3 ERS starts to disappear but the MI + MNS ERS keeps a level of 24%. Those results highlight the interest of the 8-30Hz frequency band if we seek to discriminate C3 and C4.In order to verify that a MNS is useful as a trigger to detect a movement intention, we decided to compare the classification score obtained for the traditional MI vs Rest class and our MI + MNS vs. MNS class.We pre processed our data in the following manner: (a) the frequency band is restricted to 8–30Hz; (b) we consider only the premotor frontocentral, primary motor cortex, and somatosensorial central and occipital electrodes; and (c) the classification time window is [–0.5 to 2.5 s] for MI+MNS vs. MNS, [0.5–3 s] for MI vs. Rest. These values are based on the existing literature for MI-based BCI. The average classification accuracies between a MI and a rest period, and between MI + MNS and MNS were computed for 4 different classifiers , while a passive BCI device would analyze the ERD and ERS modulations of the motor cortex to see if the patient intends to move or not. In case of such BCI could detect a modulation suggesting an intention of movement, the anesthesiologist could therefore adjust the doses of anesthetics.Our study was conducted on non-anesthetized subjects, and as shown by Blokland et al., we can expect some difference in the cerebral activity behavior once propofol is used . Moreover, the implementation of methods that depend on data coming from the same BCI session might be hard to implement in clinical settings.A preliminary analysis, presented in In future works, we intend to address such issue by using transfer learning approaches such as Riemannian geometry based methods , but in a real surgery, the MNS would intervene at different times and the cerebral activity could be modulated differently.In this study, we verified that median nerve stimulation modulates the motor cortex by first generating an ERD during stimulation and then an ERS post-stimulation. In addition, we discovered a new Post-Stimulation Rebound ERS which appears 250 ms after the stimulation in the mu and low beta band. Median nerve stimulation combined with the intention to move, i.e., the MI, has a significant impact on the ERD and ERS generated by the MNS. Indeed, despite the fact that the ERD was unaltered, the PSR is almost abolished and the rebound in the beta band is diminished. Those differences have resulted into a high accuracy classification. With these findings, we show that a BCI based on MNS is more effective than a BCI based on a MI state vs. rest. This innovative approach may improve the detection of intraoperative awareness during general anesthesia.This study was carried out in accordance with the recommendations of COERLE ethic committee of INRIA with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the COERLE ethic committee.SR and PR conceived, designed, and performed the experiments. SR, PR, NG, DS, and LB analyzed the data, contributed reagents, materials, analysis tools, prepared figures and/or tables, authored or reviewed drafts of the paper, approved the final draft.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mechanical failure in epoxy polymer and composites leads them to commonly be referred to as inherently brittle due to the presence of polymerization-induced microcrack and microvoids, which are barriers to high-performance applications, e.g., in aerospace structures. Numerous studies have been carried out on epoxy’s strengthening and toughening via nanomaterial reinforcement, e.g., using rubber nanoparticles in the epoxy matrix of new composite aircraft. However, extremely cautious process and functionalization steps must be taken in order to achieve high-quality dispersion and bonding, the development of which is not keeping pace with large structures applications. In this article, we report our studies on the mechanical performance of an epoxy polymer reinforced with graphite carbon nanoparticles (CNPs), and the possible effects arising from a straightforward, rapid stir-mixing technique. The CNPs were embedded in a low viscosity epoxy resin, with the CNP weight percentage (wt %) being varied between 1% and 5%. Simplified stirring embedment was selected in the interests of industrial process facilitation, and functionalization was avoided to reduce the number of parameters involved in the study. Embedment conditions and timing were held constant for all wt %. The CNP filled epoxy resin was then injected into an aluminum mold and cured under vacuum conditions at 80 °C for 12 h. A series of test specimens were then extracted from the mold, and tested under uniaxial quasi-static tension, compression, and nanoindentation. Elementary mechanical properties including failure strain, hardness, strength, and modulus were measured. The mechanical performance was improved by the incorporation of 1 and 2 wt % of CNP but was degraded by 5 wt % CNP, mainly attributed to the morphological change, including re-agglomeration, with the increasing CNP wt %. This change strongly correlated with the mechanical response in the presence of CNP, and was the major governing mechanism leading to both mechanical improvement and degradation. Enhance3 particles [3-filled polybenzoxazine, aluminum particle/polyester, and organo-soluble polyimide/silica material systems. Conversely, when the particle size is reduced below 100 nm, there is a clear correlation between particle size and the modulus. Polypropylene/CaCO3 composites containing smaller nanoparticles (21 nm) were shown to exhibit a higher Young’s modulus in comparison to the nanoparticles with larger size (39 nm) [However, a substantial reduction in the fracture toughness of thermoplastic polymers reinforced with rigid particulates in comparison to the as-received polymers has been reported . On the articles ,6,8. Accarticles , nanopararticles ,23. Thes (39 nm) . In cont (39 nm) , as smal (39 nm)  was studGenerally speaking, the bond equality is often attributed to process control parameters such as ultrasonication, dispersion, surface functionalization, treatment and mixing techniques. Control over numerous parameters may become an efficiency challenge (in terms of adding extra time and cost) for industrial applications, and as such, industries tend to follow straightforward mixing processes. Also, design against brittle failure  in nanocomposites is of critical importance. In a given bulk polymer material, the transition from a brittle to a ductile fracture is significant as the fracture behavior is considerably affected by the internal/external conditions. Thermoset epoxies are intensely used in structural applications despite the fact their brittleness has necessitated the incorporation of inclusions, e.g., particulate fillers. In the toughness study of nano-filled polymers, plane strain and plane stress driven cracks are superimposed to determine the impact of the incorporation on the modification of the polymer’s bulk properties, meaning that if, in the nano-filled polymer, regions with plane stress driven fracture are provoked within the space between nanoparticles, the polymer will tend to yield longer. Thereby, a macroscopic brittle matrix filled by nanoparticles may display localized plastic deformation ,28,29. AThe epoxy matrix composite was prepared by first mixing a low viscosity epoxy precursor , with Methyl nadic anhydride (MNA) that acts as a hardener with 2-Dodecenyl succinic anhydride (DDSA). Since the viscosity of the various ingredients differs, it is imperative to mix all the ingredients thoroughly. The mix bundle was stirred for a few hours before adding the curing agent. The epoxy precursor, hardeners, and curing agent were supplied by Sigma-Aldrich, Dorset, UK. 2,4,6-Tri (dimethyl aminomethyl) phenol (DMP-30) was used as the curing agent in this work. According to the epoxy specifications, the viscosity reached 60 cps which is approximately 10 % of the viscosity of conventional resins for composite applications and thus facilitated the CNP incorporation.Tg (glass transition temperature) for the final mixture was 77 °C.The final epoxy mixture was gently injected to molds suitable for the test stages  and was cured at 80 °C for 12 h in a conventional vacuum-assisted heating oven. Higher temperature curing was not used to avoid any possible drawback on the CNP-epoxy interfaces, e.g., the interfacial pre-existing dis-bond due to thermally-induced residual stresses. As per our differential scanning calorimetry (DSC) investigation, the The high-performance solvent-free polymer release agent, Zyvax® Watershield, was applied to the mold surfaces three times for easy mold release. Each time the agent was allowed to dry out on the surfaces for 30 min. The mold was then closed, and the final mixture was injected.A proportion of 1.5–2.0% of DPM-30 was added to the mixture shortly after CNPs were added and stirred for 12 h at room temperature (RT). The 1.5–2% DPM-30 enhances the strength and adds brittleness to the final mixture according to the supplier’s specifications. The temperature of the mixture was not increased before adding the curing agent in order to ensure a slower rate of cross-linking. The final mixture with the CNPs was then stirred under vacuum conditions using a Schlenk line at RT with a magnetic stirrer. The mixture was de-gassed after adding the curing agent, at <5 kPa (0.05 atmospheres).Simple short-step processing methods are highly important for industrial applications as they benefit mass production. A simple mixing method is suggested herein: The epoxy mixture was heated up to 100 °C before the CNPs were added. Heating provides a mixture with a relatively low viscosity that leads to a good CNP dispersion . CNPs, sBased on preliminary trials, the following 30-h procedure (eight stages), detailed in A mixture of approximately 30 mL epoxy was injected into an aluminum mold. The mold was cleaned using acetone, and dried using compressed air. A Zyvax® liquid sealer was first applied to protect the mold from micro-porosity, followed by application of the Watershield release agent onto the mold surfaces. Watershield was applied two or three times and was allowed to dry each time for 30 min. The CNP-epoxy mixture was then injected.Three sets of test specimens were extracted from the mold as shown in Initial investigations on the morphology and dispersion of the CNPs in cured specimens were carried out before the mechanical testing.A typical site of 2 and 5 wt % CNP filled epoxy specimens is presented in To ensure the presence of the CNPs observed in the SEM images, a quick survey of the composition was carried out in-situ using the energy dispersive X-ray spectroscopy (EDS) instrument in conjunction with the SEM. EDS data is shown in The SEM images presented a CNP dispersion with a 2D architecture. Using the phase contrast imaging capabilities of the advanced computed tomography system, VersaXRM-500  using a high-energy X-ray source (80 kV), high-resolution 3D images of the internal morphology of the nanoparticle embedded epoxy were captured. Impurities such as nanoparticles are observed in yellow (bright spots in black and white), as shown in The nanoindentation experiments were performed using a Nano Indenter G200 supplied by Agilent Technologies . The G200 uses a Nano-Mechanical Actuating Transducer (NMAT) to apply loads and measure displacements during nanoindentation tests. The load is controlled by electromagnetic actuation and the displacement is measured using a capacitance gauge. In order to characterize the specimens, indentation experiments were carried out on the specimens using the dynamic continuous stiffness measurement technique. This technique allows for continuous measurement of the contact stiffness through the indentation loading. A strain rate target of 0.05/s and a maximum depth of 5 µm was assigned to the indentations, and a Berkovich indenter pyramid tip was used. A total of 20 indentations were carried out randomly over the surface of each sample. The modulus and hardness results for 2 wt % CNP filled epoxy are presented in The morphology of the indentation site is provided in Micromechanically, stress concentration is higher surrounding a rod-shaped particle as opposed to the concentration surrounding round or elliptical particles, which will lead to a higher stress gradient upon external loading . Such a stress concentration effect rising from morphological variations from 1 to 5 wt % should have a downgrading effect on relatively large specimens. Further details are provided in In contrast to the gradual increase seen in the modulus of the 1 wt % case, see Nanoindentation tests help to realize the local mechanical response of CNPs at the micro-scale, especially surrounding CNPs, in relation to the morphologies that appear in the SEM images. Mechanical testing at the macro-scale using laboratory-scale specimens is required to obtain the properties when numerous CNPs are present. The tension and compression specimens shown in For the red dotted lines , the discrepancy was the highest between the two testing data immediately after the maximum load and stress were reached. This is attributed to the inherent toughness sensitivity (introduction of microcracks) of epoxy (and not yielding or maximum stresses) to variabilities associated with process parameters control, i.e., uncertainties. Data for the as-received and filled epoxies show a slight improvement in the compressive strength when 1 wt % is incorporated (S_(1 wt %) = 79.86 MPa, S_(0 wt %) = 79.84 MPa). An apparent increase is obtained from 2 wt % CNP inclusion (S_(1 wt %) = 91.13 MPa). This increasing trend is reversed when 5 wt % CNP is incorporated (S_(1 wt %) = 69.40 MPa). The reduction for the 5 wt % is consistent with the tensile and compressive modulus  also suggests improvements in energy absorption behavior of the epoxy in the presence of 2 wt % CNP. During the examinations, the materials and embedment procedure were constant. Therefore, adhesion properties can be assumed to be identical for all specimens. Thus, the noted improvement with the 2 wt % CNP can be assumed to be due to the smooth stress transfer between the nanoparticle and epoxy constituents.


Canonical WNT/β-catenin signaling is involved in most of the mechanisms that lead to the formation and development of cancer cells. It plays a central role in three cyclic processes, which are the cell division cycle, the immune cycle, and circadian rhythms. When the canonical WNT pathway is upregulated as in cancers, the increase in β-catenin in the nucleus leads to activation of the expression of numerous genes, in particular CYCLIN D1 and cMYC, where the former influences the G1 phase of the cell division cycle, and the latter, the S phase. Every stage of the immune cycle is disrupted by the canonical WNT signaling. In numerous cancers, the dysfunction of the canonical WNT pathway is accompanied by alterations of the circadian genes . Induction of these cyclic phenomena leads to the genesis of thermodynamic mechanisms that operate far from equilibrium, and that have been called “dissipative structures.” Moreover, upregulation of the canonical WNT/β-catenin signaling is important in the myofibroblasts of the cancer stroma. Their differentiation is controlled by the canonical WNT /TGF-β1 signaling. Myofibroblasts present ultraslow contractile properties due to the presence of the non-muscle myosin IIA. Myofibroblats also play a role in the inflammatory processes, often found in cancers and fibrosis processes. Finally, upregulated canonical WNT deviates mitochondrial oxidative phosphorylation toward the Warburg glycolysis metabolism, which is characteristic of cancers. Among all these cancer-generating mechanisms, the upregulated canonical WNT pathway would appear to offer the best hope as a therapeutic target, particularly in the field of immunotherapy. Since the discovery in 1982 of Int1 (WNT1a) as an oncogene reported in murine breast cancers, the canonical WNT/β-catenin pathway has often been found to be associated with cancer . It has The canonical WNT signaling is involved in numerous physiological processes such as embryonic development, metabolism, cell fate and epithelial-mesenchymal transition (EMT). Upregulation of the canonical WNT pathway increases the level of β-catenin in both the cytoplasm and nucleus. The dysfunction of this pathway is involved in numerous pathological processes, particularly in cancers and fibrosis –20. The There is an opposed link between the canonical WNT pathway and PPARγ  Figure . This haThe canonical WNT signaling plays a central role in the cell division cycle, particularly because the two nuclear β-catenin targets CYCLIN D1 and cMYC intervene at two key checkpoints of the cell cycle. CYCLIN D1 and cMYC act on the G1 phase and cMYC on the S phase . An osci6).Several pathways that regulate the cell proliferation act through the G1 phase . CYCLIN CYCLIN D1 is considered as key target of the canonical WNT pathway. In endothelial cells, inorganic polyphosphate induces CYCLIN D1 synthesis via activation of the mTOR/WNT/β-catenin pathway . CYCLIN A complete oscillating model of cyclins and cyclin-dependent kinases has been proposed by Gérard and Goldbeter . In the The immune cycle  is defin+ T cells and activates CD8+ effector T cells  cell response , 92. Thepression , 94.+ T cell infiltration  and myofibroblasts  play a kIn activated cancer stroma cells, there are neo-formations of contractile stress fibers containing α-smooth muscle actin (α-SMA) and non-muscle myosin (NMMIIA). These cells are named myofibroblasts. In myofibroblasts, there is co-existence of fibroblastic morphological characteristics, such as a well-developed endoplasmic reticulum and α-SMA forming contractile actin microfilament bundles. Proto-myofibroblasts are low contractile cells and when mechanical stress and TGF-β1 are present, they can differentiate into myofibroblasts that express α-SMA and NMMIIA, which are incorporated in stress fibers. This renders myofibroblasts highly contractile , 107.In cancers, mechanical and chemical conditions generating myofibroblasts promote the cancer progression. The stromal extracellular matrix (ECM) contains several molecules such as collagen, elastin, fibronectin, hyaluronic acid, proteoglycans, glycoproteins etc. The cancer stroma presents several similarities with skin healing , 109. InCancer cells release microparticles derived from the plasma which contribute to activate tumor-associated myofibroblasts. Cancer exosomes differentiate fibroblasts into myofibroblasts . In cancWith mechanical stress, TGF-β1, the most important myofibrogenic growth factor, plays a central role in myofibroblast activation and by upregulating the canonical WNT signaling , 3. In aTGF-β1 is synthesized together with the latency-associated peptide (LAP) that is cleaved in the cell and linked to TGF-β1. This leads to the small latent complex (SLC) . In compin vivo tumor growth  is often stimulated in human cancers . AKT actUpregulation of the PI3K-AKT signaling is frequently associated with cancers, such as glioblastomas, breast, pancreatic, and ovarian cancers . AKT mRNThe Warburg effect does not take into account the metabolic interactions between cancer cells and the microenvironment , 185. ThCRs are endogenous rhythms that last ~24 h, due to a negative feedback produced by a protein acting on the expression of its own gene , 204. InFrom a thermodynamic viewpoint, CRs operate far from equilibrium and are dissipative structures , 60, 217in vivo and increases the canonical WNT pathway activity (Chronic inflammation contributes to cancer development , 220. Thactivity . Conversactivity . PGE2 upSeveral intracellular processes involved in cancers are similar to those encountered in fibrosis, and particularly an activation of the canonical WNT /TGF-β1 signaling . SimilarIt is currently well-demonstrated that the canonical WNT signaling intervenes at every stages in development of many cancers. When activated, this signaling induces and initiates several periodic or oscillating phenomena, such as the cell division cycle, the immune cycle, numerous circadian rhythms, and phosphofructokinase oscillations that are dissipative structures. This is because β-catenin modifies the expression of a considerable number of genes involved in one or others of these decisive areas of the cancer pathogeny. Thus, CYCLIN D1 and cMYC influence the cell division cycle. TGF-β1 also plays an important role activating the canonical WNT signaling directly or indirectly, and by acting on both the PI3K-AKT and GSK-3β pathways. Activation of the canonical WNT signaling affects many enzymes or transporters, resulting in Warburg glycolysis, which diverts pyruvate metabolism to lactate, a useful pathway for protein synthesis in tumor cells. All these different functions of the canonical WNT give it an eminent status as target immunotherapies against cancer.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Activation of Wnt signaling induces Connexin43 (Cx43) expression via the transcriptional activity of β-catenin, and results in the enhanced accumulation of the Cx43 protein and the formation of gap junction channels. In response to Wnt signaling, β-catenin co-localizes with the Cx43 protein itself as part of a complex at the gap junction plaque. Work from several labs have also shown indirect evidence of this interaction via reciprocal co-immunoprecipitation. Our goal for the current study was to identify whether β-catenin directly interacts with Cx43, and if so, the location of that direct interaction. Identifying residues involved in direct protein–protein interaction is of importance when they are correlated to the phosphorylation of Cx43, as phosphorylation can modify the binding affinities of Cx43 regulatory protein partners. Therefore, combining the location of a protein partner interaction on Cx43 along with the phosphorylation pattern under different homeostatic and pathological conditions will be crucial information for any potential therapeutic intervention. Here, we identified that β-catenin directly interacts with the Cx43 carboxyl-terminal domain, and that this interaction would be inhibited by the Src phosphorylation of Cx43CT residues Y265 and Y313. Gap junctions are intercellular channels that permit the passage of ions, small metabolites, and signaling molecules between neighboring cells . They arThe CT domain is involved in regulating the trafficking of connexons to and from the plasma membrane, as well as the level of gap junction intercellular communication via a number of post-translational modifications and interactions with protein partners ,9,10,11.β-catenin is a critical protein in the canonical Wnt signaling transduction cascade. β-catenin (781 amino acids) consists of a well-structured central region made up of 12 armadillo repeats that are flanked by intrinsically disordered N-terminal and C-terminal domains ,31. β-ca15N-heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) experiment using purified 15N-lableled Cx43CT (V236–I382) and unlabeled full-length β-catenin , and is phosphorylated by casein kinase I), a well-structured central armadillo repeat domain , and a disordered C-terminal domain  ,40. To d-catenin A, top. ET domain . These c1H ppm) that was previously seen in the β-catenin CT domain 15N-HSQC [15N-Cx43CT residues as full-length β-catenin (DK). The decrease in signal intensity caused by increasing the β-catenin CT domain concentration was fit according to the nonlinear least-square method (DK was determined to be 210 μM (±90 μM). A surface plasmon resonance (SPR) experiment confirmed the NMR results . The N-terminal domain is the primary locus for Wnt signaling (GSK3β/E3 ubiquitin ligase), and armadillo repeat domains have been well characterized for binding partners involved in cell adhesion. Moreover, most of the binding partners to the C-terminal domain occur in the nucleus, thus potentially leaving the CT domain free to associate with a different set of proteins in the cytoplasm . Upon pu15N-HSQC . The add-catenin B, top. T-catenin B, bottome method B, inset. results . When thresidues C. Of not15N-labeled Cx43CT single or double phosphomimetics  and different concentrations of either an unlabeled β-catenin CT domain  has been overlaid with spectra when either the β-catenin CT domain or full-length β-catenin (red) were added at a single molar ratio. For the single phosphomimetic construct, among the three areas where the β-catenin CT domain interacted with Cx43CT wild type (WT), binding was completely inhibited for area three (N302–R319), significantly reduced in area two (S282–N295), and mostly preserved in area one (K259–T275). A titration of the unlabeled β-catenin CT domain was performed to determine the DK. The interaction with β-catenin decreased by approximately two-fold compared to WT (DK = 341 μM vs. 202 μM)  A, inset.The Cx43CT domain binds multiple proteins, many of which have been shown to regulate Cx43 function through altering trafficking to the gap junction plaque, opening/closing gap junction channels, disassembly, and degradation . Num+, β-catenin interacts with the Cx43 gene GJA1 (contains three transcription factor 4 (TCF)/lymphoid enhancer binding factor binding sites; [+ elicited Wnt/β-catenin signaling, which increased Cx43 expression and gap junction intercellular communication in skeletal myoblasts [+, leading to the increased nuclear localization of β-catenin and subsequent Cx43 expression [β-catenin is an intracellular signal transducer in the Wnt signaling pathway that is involved in the regulation and coordination of cell–cell adhesion and gene transcription . Ai et ag sites; ) to incrg sites; . The accg sites; . Cx43 ang sites; . A similyoblasts . Additiopression . The sugpression . This obpression , and thepression . Converspression . While tpression ,59. Thispression ,59.Shaw et al. put forth a model for connexin trafficking to the plasma membrane . Cx43 olThe importance of identifying if and where a direct protein interaction occurs is in relationship to the phosphorylation of Cx43, because phosphorylation modifies the binding affinities of the Cx43 protein partners that regulate assembly, disassembly, and channel function . For exa15N-labeled) cloned into the bacterial expression vector pGEX-6P-2  was expressed and purified in 1× phosphate-buffered saline (PBS), as previously described [The rat Cx43CT (V236–I382) (or (S255–I382) for the SPR study) polypeptide  in the absence or presence of increasing amounts (up to 285 μM) of β-catenin, β-catenin CT, or β-catenin ΔCT. Data acquisition, processing, and analysis, including calculation of the dissociation constants (DK), have been previously described [NMR data were acquired at 7 °C using a 600-MHz Varian INOVA spectrometer  upgraded with a Bruker Avance-III HD console  and outfitted with a Bruker z-axis PFG “inverse” triple-resonance cryogenic (cold) probe (Bruker). Gradient-enhanced two-dimensional escribed ,46,69.The CD experiment was performed on a JASCO J-815 CD spectrometer  at 7 °C in the far UV (260–190 nm). Spectra of the β-catenin CT were collected in 1× PBS at pH 7.4, with a 0.1-mm path length quartz cell, using a bandwidth of 1 nm, an integration time of 1 s, and a scan rate of 50 nm/min. The final spectrum was obtained from the average of five scans. All of the spectra were corrected by subtracting the solvent spectrum acquired under identical conditions. CD data were processed and converted to mean residue ellipticity using Spectra Analysis from the Jasco Spectra Manager software, Version 2.05.01 .The SPR experiments were performed on a Biacore  1000 at 25 °C. The Cx43CT (S255–I382) was immobilized onto a CM5 sensor chip by amine coupling, and the flow cell was equilibrated with the reaction buffer at a flow rate of 5 μL/min . Then, 5 μL of either the β-catenin CT (4 μM), the Cx43EL1 , or the Cx43CT  were injected over the chip, and the responses were recorded as resonance units (RU).


By computing waveforms at a period > 6 s for a wide range of 1‐D and 3‐D Earth structures, we illustrate that a time shift  between SV and SH may appear in purely isotropic simulations. This may be misinterpreted as shear wave anisotropy. For near‐surface earthquakes, apparent shear wave splitting can result from the interference of S with the surface reflection sS. For deep earthquakes, apparent splitting can be due to the S wave triplication in D′′, reflections off discontinuities in the upper mantle, and 3‐D heterogeneity. The wave effects due to anomalous isotropic structure may not be easily distinguished from purely anisotropic effects if the analysis does not involve full waveform simulations.Observations of shear wave anisotropy are key for understanding the mineralogical structure and flow in the mantle. Several researchers have reported the presence of seismic anisotropy in the lowermost 150–250 km of the mantle (i.e., D SH‐SV splitting for 1‐D and 3‐D isotropic Earth modelsSimulations of wave propagation through the lowermost mantle display apparent S and reflected wavesThe apparent splitting is due to phase interference between Anomalous Vs gradients, discontinuities, and heterogeneities may lead to misinterpretation of wave splitting Shear wave splitting up to 5 s, as reported in numerous studies, corresponds to radial anisotropy ξ=VSH2/VSV2 up to 1.06, depending on epicentral distance  varying the thickness and radial shear velocity gradient in D′′; (ii) considering a shear velocity discontinuity at the top of D′′; and (iii) including large‐scale 3‐D shear velocity variations in the mantle.In this work, we expand on previous studies by investigating the effect of the earthquake source depth on waveforms and by exploring apparent splitting for a wide range of 1‐D and 3‐D isotropic structures for waveforms at periods longer than about 6 s. We study how wave interference affects the waveforms of 2We compute synthetic seismograms using Gemini (GEM) . For completeness, we also measure tSV−tSH by using a cross‐correlation approach. Although cross–correlation measurements are more objective, they may be problematic in cases of waveform dissimilarity and differences in frequency content  between 33.1T∼ 6 s, we first validate our calculations by reproducing some of the results of Komatitsch et al.  and SHdiff waveforms (on the transverse component) that reaches 1.8 s at a distance of 120°. There are small differences in the splitting estimates between our and Komatitsch et al.'s  at stations from a temporary network in Tanzania at epicentral distance of 87–91°.From here on, we compute seismic waveforms for deep earthquakes, which are typically used in shear wave splitting studies. Specifically, we use the source‐receiver path between the m Figure  measured4.1′′ can lead to apparent splitting in the same order of magnitude as reported for many high–velocity and low–velocity regions in the D′′ layer. We assume the Preliminary Reference Earth Model (PREM) attenuation structure and source parameters from the global Centroid Moment Tensor (CMT) catalog  to 2.52% (in mod11). The strength of these velocity discontinuities is similar to that reported for downwelling regions . At distances shorter than 90° when S and ScS are separated by more than 4–5 s, the SH waveforms are double peaked .The interference of S with ScS for models with a shear velocity discontinuity at the top of D′′ larger than 1.14% (models from mod9 to mod11) generates apparent splitting. The D′′ discontinuity causes, in fact, an S wave triplication comprising a direct S wave (Sab), an S wave turning below the discontinuity (Scd), and an S wave reflecting off the discontinutiy (Sbc). Their relative arrival times depend on the epicentral distance and velocity jump. For mod10  for most models of Figure The apparent shear wave splitting values measured for models mod9–mod11 are summarized in Figure 5To understand whether 3‐D velocity heterogeneity can complicate further the interpretation of shear wave splitting, we repeat some of the experiments described in sections 5.1Figure SV at a distance of 90° and moves out with distance. This signal arrives about 20 s after S at 96°. Travel‐time calculations show that this strong arrival is consistent with the arrival time of S∧220P, the S wave reflected off under the 220‐km mantle discontinuity. The interference between S and S∧220P has a minor effect on the waveforms at azimuth of 270° because of the higher amplitude ratio between S and S∧220P. Thus, the difference in waveforms and apparent splitting observed at the two azimuths is due to the focal mechanism used in this experiment that radiates seismic energy differently along the two azimuths analyzed.Figures S wave modeled in the 3‐D Earth models . In our study, the differences in Vs structure near the CMB of the two 3‐D Earth models compared to the PREM model lead to distinct CMB conditions and thus possibly to the observed differences in waveforms and shear splitting values of the diffracted waves.For an azimuth of 270°, the  Figures g and 8h d et al.  attribut5.2Mw 5.8 Banda Sea earthquake. We superimpose the isotropic part of the global model SGLOBE‐rani  is located at the top of the D′′, the interference of S triplicated at the discontinuity and ScS may lead to apparent splitting up to 7 s depending on the strength of the discontinuity and epicentral distance.We have found that in most of the cases apparent splitting is due to the anomalous interference of the direct h et al. , and epiS but also the epicentral distance at which S starts to diffract along the CMB.We also found that 3‐D Earth structure can modify the waveforms and enhance or reduce the apparent splitting. In fact, seismic heterogeneity affects not only the arrival time and waveform of the waves interfering with the direct VSH>VSV) has been detected in several high D′′ shear velocity regions underlying present or past subduction zones, such as beneath the Caribbean, Alaska, and North Siberia  used approximate forward modeling schemes to show that the distinction between the effects of isotropic and anisotropic structure on the Sdiff waveforms is not trivial. Komatitsch et al. (S due to finite frequency effects and attributed the Sdiff apparent splitting to the different sensitivity of SV and SH to the boundary conditions between the solid mantle and liquid outer core. In particular, they highlighted that apparent shear wave splitting can result from the misidentification of triplicated phases, which is compatible with our results. In addition, Kawai and Geller (SV shear waves very close to the CMB is inherently limited due to the boundary condition of zero tangential traction at the CMB. In this work we confirm the apparent splitting reported by these previous studies and we emphasize phase interference as being a key cause of apparent splitting. Moreover, our study also complements previous work by showing that 3‐D Earth structure can either enhance or reduce apparent splitting, depending on the region through which the waves propagate, which adds complexity to the shear wave splitting analysis. Nevertheless, we highlight that in the case of S waves diffracted along the core, phase interference and different sensitivity to the CMB can both cause apparent splitting.This study follows previous studies that highlighted the possibility of apparent h et al.  used theh et al.  considerh et al.  studied d Geller  showed t′′ thickness, discontinuities, and 3‐D heterogeneity. Our analysis highlighted a strong interplay between the various source and structure parameters considered, which may lead to a misinterpretation of the splitting and potentially erroneous constraints on intrinsic D′′ anisotropy. Full waveform modeling considering realistic sources and a wide range of 1‐D and 3‐D Earth models as in this study is a promising way to address these issues. While the illustrative examples based on the global smooth 3‐D Earth models used in this study are a useful first step to quantify their effect on apparent shear wave splitting, future efforts will be directed toward comprehensive 3‐D full wavefield analyses including more complex D′′ structures and multiple source‐receiver orientations.In conclusion, we systematically quantified apparent shear wave splitting for several source depths and for a wide range of Earth models, including anomalous Vs gradients, DSupporting Information S1Click here for additional data file.


Background: Activation of macrophages and infiltration are key events in acute liver injury (ALI). Kv1.3 plays an important role in regulating immunologic functions of macrophages and is extensively recognized as a potential ion channel for immunological diseases.Objective: We hypothesized that blockage of Kv1.3 may influence ALI by inhibiting macrophages infiltration in damaged liver tissues.Methods: Margatoxin was administered into the peritoneal cavity of ALI mice. The impact of this treatment on ALI and macrophage migration in vivo and in vitro was determined using immunohistochemistry, transwell migration, and wound healing assays.Results: MgTX treatment alleviated ALI in mice, as evidenced by reduced macrophage infiltration in liver tissues and lower serum levels of liver ALT and AST. RNA-seq profiling analysis showed that the most obvious change by MgTX treatment was downregulation of δ-catenin, a protein known to be associated with macrophage migration. The effect of MgTX on macrophage migration and involvement of δ-catenin was confirmed by transwell and wound healing assays. Overexpression of δ-catenin in RAW264.7 cells promoted migration, an event that was suppressed upon silencing of δ-catenin. Mechanistically, the expression of RhoA was regulated by the overexpression or knockdown of δ-catenin.Conclusion: These findings suggest a role for blockage of Kv1.3 channel in macrophage migration and reveal a new target in the treatment of ALI. Infiltration of monocytes plays a crucial role in the progression of neuroinflammatory diseases, inhibit monocytes infiltrate into injury CNS recognized as a potential therapeutic strategy Acute liver failure (ALF) is caused by serious hepatic injury with abnormal function of hepatocyte and leading to different clinical syndrome, such as coagulopathy, encephalopathy, and circulatory dysfunction. ALF is related with high mortality in liver diseases, ranging from 30 to 80%3Centruroidesn margaritatus. It is stabilized by three disulfide bridges with a molecular weight of 4185. Margatoxin has a special affinity for Kv1.3 and is a high selective inhibitor for Kv1.3 channelKv1.3 belongs to a subtype of voltage-gated potassium channel, which has six transmembrane domains S1-S6), including a voltage sensor (S4) and a pore-forming region, includiWe established an ALI mouse model using LPS+D-GAIN  and treated these mice with MgTX injection in the peritoneal cavity. The effect of MgTX on macrophage migration into liver tissues and alleviation of liver injury was investigated. RNA-seq analysis showed that MgTX inhibited macrophage migration through a decrease in δ-catenin RNA expression. Furthermore, we found that δ-catenin could modulate the expression of its downstream protein RhoA to inhibit macrophage migration and, ultimately, alleviate ALI. Our proof-of-concept study suggests that inhibition of Kv1.3 is a potential therapeutic strategy for acute liver diseases.Animal protocols were approved by the Animal Care and Use Committee of Anhui Medical University, Hefei, China. C57BL/6 20 ± 2 g) male mice were purchased from the Experimental Animal Center of Anhui Medical University for the LPS+D-GAIN model 0 ± 2 g mThe levels of alanine aminotransferease (ALT) and aspartate aminotransferase (AST) in serum were measured using ALT and AST activity assay kits . The absorbance was measured at 510 nm with a Multiskan MK3 microplate reader .Serum was extracted from blood using centrifugation  and stored at -80˚C for subsequent use. The levels of TNF-α was determined in mouse serum using ELISA kits according to the manufacturer's protocol. mouse TNF-α ELISA kit (cat. no. JYM028Mo) was used and all purchased from Jiyinmei biotechnology co., Wuhan.The middle portion of the left hepatic lobe was sectioned and fixed in 4% paraformaldehyde for 48 h, then the tissues were embedded in paraffin, and 5 μm thick slices were stained with hematoxylin - eosin (HE) for morphological analysis, Masson's trichrome for evaluation of collagen expression.2 incubator.The RAW264.7 cell lines was purchased from the Type Culture Collection of the Chinese Academy of Sciences . Cells were cultured in Dulbecco's-modified Eagle's medium  supplemented with fetal bovine serum , penicillin and streptomycin (1%) and cultured at 37°C in a 5% COTo identify effects of MgTX on monocytes migration in acute liver injury, flow cytometry analysis was performed. Fresh liver was dissociated into single-cell suspensions by combining mechanical dissociation according liver dissociation kit . Cells were incubated away from light with fluorescent-labeled anti-mouse antibodies for 15 min at 4°C, then cells were washed in PBS (1ml) and centrifugated (300 g) for 5 min, 300 µl resuspended at ~5×106/ml after 1 h for flow cytometry . FlowJo version 10 software  was used to analyze the data. Antibodies used in this experiment were as follows: anti-CD45-perCP-Cy5.5 , anti-Ly6GLy6C-PE , anti-CD11b-FITC , and CD192-AF647 .C. reinhardtii reference genome, using the TopHat2 aligner, dates can be got from Phytozome 11 database (JGI). Bioconductor edger was used for differential expression analysis of RNA-seq expression profiles. Gene absolute values of p ≤0.05 and log2 (fold change) ≥1 were set as thresholds of DGEs . After analysis, the DGEs were subjected to enrichment analysis of GO functions and KEGG pathways.RAW264.7 cells grown in DMEM medium and subjected to 10 nM MgTX for 12 h treatment were collected. Untreated cells served as control. The total RNA was extracted using standard protocols and high-throughput sequencing was detected on illumina HiSeq 2000 platform in GENE DENOVO . Raw sequences containing adapters and low-quality bases were filtered and mapped to the 2 incubator for 12 h.To overexpress and downregulate the expression of δ-catenin, RAW264.7 cells were transfected with plasmid or small interfering RNA (siRNA), respectively, using lipofectamine 2000 reagent  following to the manufacturer's instructions. SiRNA oligonucleotides against δ-catenin, overexpression plasmid was designed and constructed by Shanghai GenePharma Corporation. RAW264.7 cell lines were transfected with siRNA or plasmids in opti-MEM culture medium . After 6 h transfection, the opti-MEM culture medium was changed to DMEM, and cells were cultured at 37°C in a 5% CO5 /well using serum-free medium, and then the drug (MgTX) or an equal amount of vehicle control (distilled water) was added to the cell suspension. Next, 200 μL cell suspension was placed with serum-free medium into the upper chamber, 600 μL same medium with 10% FBS was placed in the lower chamber. After culturing for 16-18 h at 37°C in 5% CO2 incubator, the upper chambers were removed from the Transwell system and fixed in methanol for 20 min. Cells on the upper side of the filters were removed with cotton-tipped swabs and the filters washed with 0.01M PBS. Cells on the lower side of the filters were stained with 0.5% crystal violet in PBS for 15 min. The image of the cells was counted under a microscope, and the number of migrating cells was recorded with ImageJ software. Each treatment was performed in triplicate and repeated for at least three times.Transwell chambers  were pre-wet with serum-free DMEM for 30 min prior to use. The number of cells is adjusted to a concentration of 1 × 10Wound healing assay was performed as previously described δ-catenin and GAPDH were performed with ThermoScript RT-qPCR kits . GAPDH was used to normalize input RNA. Relative RNA expression level was calculated following the standard 2-ΔΔCt method. each experiment was performed in triplicate and repeated for at least three times.Total RNA was extracted from RAW264.7 cell lines using TRIzol reagent . Using ThermoScript RT-qPCR synthesis kit  to synthesized cDNA following to the manufacturer's protocol. Real-time quantitative PCR analysis for mRNA of RAW264.7 cells were lysed with protein extraction solution . Protein concentration of each sample was measured by NanoDrop 2000 Spectrophotometer . Samples were performed using electrophoresis on SDS-PAGE and transferred onto PVDF membrane . After PVDF membrane was sealed with nonfat 5% milk for 2 h, then followed by incubation with primary antibodies at 4°C overnight. The concentration of primary antibodies against δ-catenin  and β-actin were used at 1:500. Subsequently, the PVDF membranes were washed three times with TBST, followed by incubation with secondary antibodies  at room temperature for 1.5 h. After washing three times with TBST, protein blots were observed using ECL-chemiluminescent kit . P < 0.05.Statistical analysis was performed with the Statistical Package for Social Sciences v.13.0 . Each experiment was performed at least three times and the date are presented as the mean ± SD. Statistical differences of the results were analyzed by one-way ANOVA. Dates were considered statistically significant when+) from peritoneal cavity to liver tissues  and subjected to MgTX treatment. ALI and control mice were euthanized 12 h after LPS+D-GAIN or vehicle injection. Hematoxylin eosin (H&E) staining showed that LPS+D-GAIN injection caused significant ALI including hepatic steatosis, liver injury, and histological changes, which were significantly attenuated in MgTX treatment mice. In comparison, no injury was evident in the control group  . The RNA-seq analysis is shown in a volcano plot  could inhibit the expression of δ-catenin in RAW264.7 cell lines. As shown in Fig.We examined the effects of MgTX on macrophage migration by performing cells wound healing and transwell assays. Representative figures of cell migration assays were shown in Figure Figure , macrophage migration was inhibited by δ-catenin siRNA and the inhibition effect was enhanced by MgTX (10 nM) treatment. The efficiency and specificity of δ-catenin knockdown was confirmed by Western blotting in Figure . The expression of δ-catenin was downregulated by δ-catenin siRNA, and further inhibited with MgTX (10 nM) treatment. In contrast, as shown in Figure , overexpression of δ-catenin effectively enhanced the migration of macrophages, compared with control RAW264.7 cells, while this enhancement was attenuated by MgTX (10 nM) treatment. The efficiency and specificity of δ-catenin overexpression was verified by Western blotting in Figure . The expression of δ-catenin was upregulated by δ-catenin overexpression and decreased with MgTX (10 nM) treatment.We next investigated the impact of δ-catenin expression on the migration of RAW264.7 cell lines by performing wound healing and transwell assay. δ-catenin was knocked down or over expressed in RAW264.7 cell lines by transfection assays for 12 h. As shown in Figure  we found that the expression of Rho A was upregulated in δ-catenin-overexpressing cells and significantly inhibited in δ-catenin-knockdown cells. In comparison, the expression of Rac and Cdc42 had no obvious changes in response to alteration of δ-catenin expression. In further examination of the relationship between δ-catenin and Rho A, we found that the expression of Rho A was downregulated by δ-catenin loss and MgTX (10 nM) treatment, and upregulated by δ-catenin overexpression in RAW264.7 cell lines in Figure We next investigated the mechanism of δ-catenin regulation of macrophages migration and its downstream target in RAW264.7 cell lines. Previous RNA-seq results showed that Rho GTPases signal was downstream of δ-catenin. Therefore, we evaluated the expression of mRNA levels of Rho GTPases, including Rho A, Rac, and Cdc42, using qRT-PCR. As shown in in vitro. This could be the underlying molecular mechanism by which δ-catenin regulates the migration of RAW264.7 cell lines.In this study, we demonstrated that δ-catenin/Rho GTPases signaling pathway is involved in the modulation of migratory ability of RAW264.7 cells through regulating RhoA expression 2+ signaling, T cell proliferation, and IL-2 secretion high monocytes in hemolytic uremic syndromeKv1.3 is a subtype of voltage-gated potassium channel, initially identified in human T lymphocytesCentruroides margaritatusδ-Catenin gene in mice leads to severe damage in cognitive function and short-term or long-term synaptic plasticity, which is associated with learning and memory of the brain et al., detected the expression of δ-catenin in lymphatic endothelial cells, and those authors found that δ-catenin increased lymphangiogenesis in vitro and in vivo, and through modulation of small RhoGTPase activities. Consistent with inactivation of δ-catenin, lymphangiogenesis and growth of tumor metastases were inhibited in mouse modelsPrevious research and the data presented herein indicated that Kv1.3 could inhibit the macrophages migration; however, the mechanism of the Kv1.3 channel modulates macrophage migration is unclear. MgTX has 39 amino-acid-long peptide stabilized by three disulfide bridges with a molecular weight of 4185, and was extracted from the venom of the scorpion To further understand the essential role of δ-Catenin in mediating macrophage migration by RhoGTPase pathway, we investigated the impact on RhoGTPase family, which is located in downstream of δ-Catenin protein. Small Rho family of GTPase are related to cell motility, and previously studies identified that δ-catenin can regulate neuronal cell and vascular endothelial cell motility through Rho GTPaseIn summary, we demonstrate for the first time that MgTX can alleviate ALI though inhibition of macrophage migration and the δ-catenin/RhoA pathway may be an important player in mediating this process. The current findings not only increase our understanding of how MgTX inhibits macrophage migration in ALI, but also suggest a new direction for development of potential targets in acute liver injury.


Endoscopy nurse (RN) has a pivotal role in administration and monitoring of moderate sedation during endoscopic procedures. When sedation for the procedure is administered and monitored by an anesthesia specialist, the role of an RN is less clear. The guidelines on this issue by nursing and gastroenterology societies are contradictory.  Survey study of endoscopy lab managers and directors at outpatient endoscopy units in Texas. The questions related to staffing patterns for outpatient endoscopies and responsibilities of different personnel assisting with endoscopies.  Responses were received from 65 endoscopy units (response rate 38%). 63/65 (97%) performed at least a few cases with an anesthesia specialist. Of these, 49/63 (78%) involved only an endoscopy technician, without an additional RN in the room. At 12/49 (25%) units, the RN performed tasks of an endoscopy technician. At 14/63 (22%), an additional RN was present during endoscopic procedures and performed tasks not directly related to patient care.  Many ambulatory endoscopy units do not have an RN present at all times when sedation is administered by an anesthesia specialist. An RN, when present, did not perform tasks commensurate with the education and training. This has implications about optimal utilization of nurses and cost of performing endoscopies. Endoscopies at the ambulatory surgical centers (ASCs) are well-established models of care offering convenience, efficiency, and economy for low-risk endoscopies. For ASCs to be successful, it is important to keep quality and patient satisfaction high and costs low. Optimal staffing of the endoscopy units is, therefore, essential. The endoscopy team usually consists of a registered nurse (RN) who administers moderate sedation and an endoscopy technician who assists with technical procedural-related activities such as taking biopsies and removing polyps. The person performing the tasks of a technician may be another RN or a trained unlicensed person. In the last few years, more and more endoscopists prefer sedation with propofol, which is faster and shorter acting . PropofoThe American Society of Gastrointestinal Endoscopy (ASGE) guidelines state that when an anesthesia specialist is responsible for sedation , an addiWe conducted a survey of ambulatory surgical centers (ASCs) and endoscopy units in Texas, United States, to determine the practice patterns regarding type of sedation (moderate vs. deep), staffing of endoscopy units, and the role of RNs during procedure when sedation is administered by an anesthesia specialist.https://www.surveymonkey.com/. The questions asked included type of endoscopy unit ; number of endoscopic procedures performed per month; type of procedures; percentage of patients with different American Society of Anesthesiologists (ASA) classification; method of sedation; and personnel present in the endoscopy room during procedure and their responsibilities during procedure.A questionnaire was constructed after open-ended interviewing of gastroenterologists and nurses from two institutions. The responses were reviewed by additional gastroenterologists and nurse managers from three different institutions, not included in the study, for linguistic, internal, and external validation of the questionnaire. An online version of the survey was created using The names of ambulatory surgical centers performing endoscopies were obtained from the Texas Department of State Health Services . An emaiData were summarized using descriptive statistics. Response rates were calculated as number of surveys with responses divided by number of surveys distributed. All the data supporting the results are shown in the paper and can be applicable from the corresponding author.The surveys were distributed to 172 endoscopy units and responses received from 65 (response rate 38%). Details of the ASCs are shown in Overall, 63/65 (97%) of endoscopy units performed cases with an anesthesia specialist. Of these, 30 (46%) endoscopy units performed 100% of their procedures and 15 (23%) performed 75–99% of their procedures with deep sedation administered by an anesthesia specialist. Two endoscopy units performed their cases only with moderate sedation and did not use an anesthesia specialist.Of the endoscopy units that performed their cases with an anesthesia specialist, 49/63 (78%) involved only an endoscopy technician, without an additional RN in the room. At 12/49 (25%) endoscopy units, the RN performed tasks of an endoscopy technician. The other endoscopy units had a “floating nurse” who was available, if needed.At 14/63 (22%), an additional RN was present during endoscopic procedures and performed tasks not directly related to patient care. The responsibilities of RNs included at least one of the following: performing a time out before the procedure, documenting endoscopic accessories used, and documenting/labeling pathology samples. At some units, RNs had additional responsibilities. For example, at 7/26 (27%) endoscopy units, the RNs helped with consents before the procedures. At 2/26 (8%) endoscopy units, the RNs completed the preliminary endoscopy report or discharge instructions after discussions with the endoscopist.Twenty-six endoscopy units had at least one RN present during endoscopy, when sedation was administered by an anesthesia specialist (12 endoscopy units had 1 RN and 14 units had 2 RNs). The reasons given by endoscopy units for having an RN along with anesthesia specialists included regulatory requirement to have an RN in the room , complete documentation requirements , improved patient safety , and need for an RN for submucosal injections . At the 37 endoscopy units without an RN, submucosal injections were mainly performed by the technician assisting with the procedures except at 7 (19%) units where a floating RN was called, as needed. At two endoscopy units, the technician was allowed to assist with submucosal injection of saline and dye but not diluted epinephrine.ASCs avoid urgent circumstances and high-risk patients and are especially well suited for care-process standardization. Reducing variation and standardizing workflows that allow staff to work at their level of training and scope of practice is necessary to provide high quality, cost-effective care. Conflicting statements from professional societies or ambiguous statements from credentialing and regulatory bodies can create confusion about standards of practice. We highlight such an issue in our study, which although focused on endoscopy is applicable to other procedures at ASCs.What should be the appropriate staffing during endoscopy when endoscopy sedation is provided by an anesthesia specialist? What should be the role of an RN in this situation? The American Society of Gastrointestinal Endoscopy states, “When endoscopy is with an anesthesia specialist, one endoscopy staff member is required to assist the endoscopist with the technical portion of the procedure. This person may be an unlicensed active personnel (UAP), licensed practical nurse (LPN) or an RN. The presence of an RN is not mandatory in this setting” , 6. The The issue highlighted in our study, while focused on endoscopy, has a more generalized appeal for other procedures at ASCs. Conflicting statements from professional societies or ambiguous statements from credentialing and regulatory bodies are not uncommon. Our survey is the first study, to our knowledge, to determine how ASCs choose to staff their endoscopy units and the rationale for their approach. Our results show that, when an anesthesia specialist administers sedation, less than one fourth of the ambulatory endoscopy units have an RN present during the procedure. Most of them instead have a “floating” nurse who is available to help as needed. Notably, the change in staffing model of not having an RN in the room appears to be a recent trend. ASGE survey from 2013 had reported that in ambulatory endoscopy units, an RN was present in the room along with anesthesiology provider 62% of the time . We specThe responsibilities of an RN, when present in the room along with an anesthesia specialist, mainly included performing a surgical time out, documenting endoscopic accessories used (for billing purposes), and/or helping with pathology samples—none of which require a person to have nursing knowledge or expertise. When an RN was not present, these tasks were completed by the endoscopy technician and/or anesthesia specialist. The increasing documentation requirements and paperwork sometimes necessitates an additional person in the room, other than a technician. Some endoscopy units used the floating RN or another technician for these tasks.One of the arguments of having an RN in the room along with the anesthesia specialist is increased patient safety. The premise of such an argument would be that deep sedation by an anesthesiologist has more complications compared to moderate sedation by an RN and that an extra RN is needed helpful to respond to these complications. There is no data to support these premises. Before the current regulation that administration of propofol can only be performed by an anesthesia specialist, RNs administered propofol under the endoscopist's supervision. This was referred to as nurse-administered propofol sedation (NAPS), the safety of which has been well established in many prospective and retrospective studies , 10. NotAnother reason why endoscopy units need RNs during the procedure is the belief that regulations require that submucosal injection of saline, epinephrine, and dyes into the gastrointestinal wall can only be performed by a nurse and not an unlicensed personnel (UAP) or a technician. The regulations on what licensed and unlicensed personnel are permitted to do during a procedure are determined by the state but the directives are often not stated clearly. The Joint Commission, the Accreditation Association for Ambulatory Health Care, and Centers for Medicare and Medicaid Services do not define the specific qualifications or number of staff required –13. RathThe question if an RN should be present at all times during the procedure is important and especially relevant in present health care scenario where there is severe shortage of RNs and endoscopy units are under pressure to provide more cost-efficient care to the patients. A recent report by American Association of Colleges of Nursing estimates the nursing shortage to be 260,000 by year 2025 . In our When an anesthesia specialist administers sedation, RNs present in the room did not perform tasks commensurate with their training and experience. Endoscopy nurses are a valuable resource and their knowledge and expertise can be utilized in more important ways. For example, the endoscopy nurses can be helpful in tracking quality indicators of endoscopy. The endoscopy nurse can also help improve communication about endoscopic procedures with the patients. In a recent study, patients at a safety-net hospital, who gave informed consent for an endoscopy after an educational class given by nurses, reported being better informed about the procedure compared to patients who were consented in the gastroenterology clinic .Our study has limitations. First, the study was conducted in the United States and may not be applicable to endoscopy units in other countries, which have different regulatory requirements. Second, our study population was restricted to ambulatory endoscopy units, which affects generalizability of the results to endoscopy units within a hospital. This was intentional to ensure homogeneity of patient population across different endoscopy units. For example, the patients were mostly ASA I and II and less likely to include complex indications. Second, we surveyed only endoscopy units in the state of Texas. We did so to avoid regional variations in policies and regulations regarding scope of practice for surgical technicians, LPNs, and RNs. Fourth limitation is the possibility of information bias. Response bias could have occurred if endoscopy units that do not use an RN in the endoscopy unit were more likely to respond. However, we believe that our data is more representative of the current trends than nonresponse bias. A 2012 survey of endoscopy practices conducted by ASGE showed that nationwide 66% endoscopy units use propofol for sedation which is very similar to our data. Even, if there is some response bias, our survey shows the feasibility and safety of procedure with an anesthesia specialist without an RN in the room. Finally, our survey does not address the issue of safety of endoscopic procedure with different staffing models.In conclusion, our study highlights different endoscopy staffing models and how different endoscopy units are adapting to the changes in health care—increasing demand for the more expensive deep sedation, decreasing reimbursements and nursing shortage. Interests and safety of the patients come first and endoscopy units should ensure that the staff assisting with the procedures are well trained. Most ambulatory endoscopy units now do not have an RN present at all times when sedation is administered by an anesthesia specialist. This staffing model meets the regulatory requirements in the US, is not associated with any adverse outcome, and may allow more appropriate utilization of endoscopy nurses.