Wnt has diverse regulatory roles at multiple cellular levels and numerous targeting points, and aberrant Wnt signaling has crucial roles in carcinogenesis, metastasis, cancer recurrence, and chemotherapy resistance; based on these facts, Wnt represents an appealing therapeutic target for cancer treatment. Although preclinical data supports a role for the Wnt signaling pathway in uterine carcinogenesis, this area remains understudied. In this review, we identify the functions of several oncogenes of the Wnt/β-catenin signaling pathway in tumorigenesis and address the translation approach with potent Wnt inhibitors that have already been established or are being investigated to target key components of the pathway. Further research is likely to expand the potential for both biomarker and cancer drug development. There is a scarcity of treatment choices for advanced and recurrent endometrial cancer; investigating the sophisticated connections of Wnt signaling networks in endometrial cancer could address the unmet need for new therapeutic targets.This review presents new findings on Wnt signaling in endometrial carcinoma and implications for possible future treatments. The Wnt proteins are essential mediators in cell signaling during vertebrate embryo development. Recent biochemical and genetic studies have provided significant insight into Wnt signaling, in particular in cell cycle regulation, inflammation, and cancer. The role of Wnt signaling is well established in gastrointestinal and breast cancers, but its function in gynecologic cancers, especially in endometrial cancers, has not been well elucidated. Development of a subset of endometrial carcinomas has been attributed to activation of the APC/β-catenin signaling pathway (due to β-catenin mutations) and downregulation of Wnt antagonists by epigenetic silencing. The Wnt pathway also appears to be linked to estrogen and progesterone, and new findings implicate it in mTOR and Hedgehog signaling. Therapeutic interference of Wnt signaling remains a significant challenge. Herein, we discuss the Wnt-activating mechanisms in endometrial cancer and review the current advances and challenges in drug discovery. The human endometrium is a proliferative, angiogenic, and dynamically regenerated normal tissue that is the anatomic prerequisite for pregnancy. Stem/progenitor cells  likely contribute to both the rapid endometrial cyclic growth and regeneration of 4–14 mm of mucosa per cycle during a woman’s reproductive lifespan and endometrial regeneration/repair postpartum. During the reproductive life of women, the human endometrium undergoes dynamic cyclical changes with ~400 cycles of regeneration, differentiation, and shedding (menstruation). These cycles are regulated by many signaling pathways but primarily by estrogen and progesterone hormone signals ,3. TumorEndometrial cancer is the sixth most common gynecologic malignancy of the female reproductive organs and, while rates vary by geographic region, overall, it afflicts approximately 27.8 per 100,000 women worldwide annually . In the Genetic alterations as well as various cell-signaling pathways have been implicated in endometrial cancer development and progression. These pathways include Wnt/β-catenin signaling cascades , the PI3K/AKT/mTOR signaling pathway, the MAPK/ERK pathway, the ErbB signaling pathway, the VEGF/VEGFR ligand–receptor signaling pathway, and the p53-P16INK4a signaling pathway. Endometrial tumor tissues have been shown to contain mutations in these signaling pathways, which are generally regarded as primary drivers of carcinogenesis . A betteThe Wnt/β-catenin signaling pathway is an extremely conserved pathway that is involved in a variety of cellular processes in the female genital system, including development, cell proliferation, cell survival, adhesion, and motility as well as the regulation of the menstrual cycle. In addition to its crucial role in tissue homeostasis, aberrant Wnt signaling plays a significant role in many diseases from cancer to metabolic disorders ,15,16,17Early studies into the role of Wnt signaling in endometrial cancers mainly focused on identifying β-catenin gene mutations. However, mutation of APC (Adenomatous polyposis coli) or CTNNB1 (the gene that encodes β-catenin) is rare in endometrial cancer, indicating that other mechanisms are responsible for the aberrant activation of β-catenin ,25,26,27Unlike for other cancers, the mechanism of Wnt signaling participation in endometrial cancer has not been elucidated and is not limited to the involvement of β-catenin and APC mutations. In this review, we present an overview of the Wnt signaling pathway and its activating mechanisms in endometrial cancer. We will address the common Wnt pathway-associated mutations identified in endometrial cancer and will further review the current therapeutic options targeting Wnt signaling considering both their potential and their limitations.The Wnt signaling pathway is evolutionarily highly conserved and is a crucial cascade regulating development and stemness. Wnt signaling is also firmly associated with several cancers. This signaling network can be divided into two modes based on the role of β-catenin: the β-catenin-dependent pathway is called “canonical Wnt/β-catenin signaling” and the β-catenin-independent pathway is called the “noncanonical pathway.” The noncanonical Wnt/β-catenin pathway can be further subdivided into the planar cell polarity (PCP) pathway and the Wnt/Ca2+ pathway ,42,43 F.In both the canonical and noncanonical pathways, signaling is initiated by binding of Wnt ligands to the extracellular cysteine-rich domain (CRD) at the amino terminus of Frizzled receptors (Fzd) and a number of recently linked coreceptors, including receptor tyrosine kinase-like orphan receptor 1 (ROR1), receptor tyrosine kinase-like orphan receptor 2 (ROR2), and receptor-like tyrosine kinase (Ryk). This ligand–receptor interaction activates canonical WNT/β-catenin and noncanonical WNT/PCP and WNT/Ca2+ signaling pathways. This mechanism is interfered with by several inhibitors and gatekeeper molecules, including the families of SFRPs and DKKs ,44,45.JUN, c-Myc, CCND1(Cyclin D1), and AXIN2 among others [The canonical Wnt/β-catenin pathway is activated by binding of Wnt to a transmembrane receptor complex that is formed from the seven-pass transmembrane Fzd and the co-receptor low-density lipoprotein receptor-related protein 5 or 6 (LRP5/6); binding is enhanced by the R-spondin/Lgr interaction. This Wnt–Fzd–LRP6 complex recruits scaffolding protein Dishevelled (Dvl), which leads to the phosphorylation of LRP6 and recruitment of the Axin complex to the receptors. This signaling cascade hinders Axin-mediated β-catenin phosphorylation and subsequently stabilization of β-catenin. Liberated β-catenin then accumulates and translocates to the nucleus where it binds to T cell factor and Lymphoid enhancer-binding factor 1 (TCF/LEF) and promotes the transcription of Wnt target genes, such as g others .As mentioned above, the β-catenin-independent or noncanonical pathway can be further divided into two different branches, the planar cell polarity (PCP) pathway and the Wnt/Ca2+ pathway, both of which are activated by Wnt. The PCP pathway regulates cell motility and polarity through the activation of small GTPases, RhoA, Rac, and the c-Jun N-terminal kinase (JNK). The PCP pathway can be demonstrated by Wnt–Fzd interaction; however, receptor tyrosine kinase-like orphan receptors (RORs) like ROR1, ROR2, and receptor tyrosine kinase (Ryk) can also serve as Wnt receptors to activate β-catenin-independent pathways ,48,49,50Wnt/β-catenin pathway activation is a pivotal regulator of epithelial-mesenchymal transition (EMT) ,52,53. WThe role of aberrant Wnt signaling in cancer was first discovered during tumor induction in a mouse model following proviral integration at the WNT1 locus ,56. In rAPC and CTNNB1 (β-catenin) genes that lead to hyperactivation of the pathway. Mutations of β-catenin that result in Wnt signaling dysfunction have frequently been observed in many other cancers, including endometrial cancer, ovarian cancer, medulloblastoma, hepatocellular carcinoma, and Wilms’ tumor, with many studies supporting the idea that elevated β-catenin levels are associated with poor prognosis [It is very well reported that aberrant activation of the canonical Wnt/β-catenin signaling pathway is associated with the development of colorectal cancer through mutations in the rognosis ,57.Furthermore, β-catenin is not the only important component of the Wnt signaling network. Several studies have been published in the last decade that correlate changes in Wnt ligands, receptors, gatekeepers, and downstream effector molecules to almost every type of human tumor. Dysregulation of both canonical and noncanonical Wnt signaling has been shown in numerous cancers ,61,62,63Endometrial cancer is the most common gynecologic malignancy found in women worldwide and it is the second leading cause of gynecologic cancer death in the United States ; for 202Increasing age, long-term use of unopposed hormone therapy, a history of nulliparity or infertility, irregular menstrual cycles, polycystic ovarian syndrome, obesity, diabetes mellitus, and hypertension are all risk factors for endometrial carcinoma ,96,97,98The majority of endometrial carcinomas are produced from endometrial glands (adenocarcinomas), while the remainder are derived from the supporting stroma (sarcomas) . EndometThe second most common type of endometrial cancer is the serous subtype. These tumors are generally diagnosed at a high grade, are poorly differentiated with deep invasion into the underlying uterine wall (myometrium), are very aggressive, and have poor outcomes. They generally do not express the receptors for estrogen or progesterone and are not responsive to hormonal treatment. In the United States, serous endometrial cancers are more common in African American women. The 5-year survival rate for serous cancers is 53% ,102. AnoCTNNB1 (β-catenin), PIK3CA, and KRAS, along with microsatellite instability and POLE mutations  project, which revealed key molecular pathways and mutations associated with endometrial cancers; endometrioid subtypes most frequently had mutations of PTEN (a protein and lipid phosphatase), utations  whereas cadherin ,107,108.APC, Axin, CTNBB1, Wnt ligands, Wnt inhibitors Dickkopf3 (DKK3) and Dickkopf1 (DKK1), or the secreted Frizzled-related proteins may be a key driving event in the development of endometrial cancer. Nuclear accumulation of β-catenin  is the predominant alteration found in endometrioid tumors, but only a few studies have examined the role of Wnt signaling in its etiology [Abnormal Wnt/β-catenin signaling plays an important role in endometrial cancer onset. Around 40% of endometrial cancers (mostly endometrioid histology) show abnormalities in the Wnt signaling pathway. Activation of the Wnt signaling pathway via alteration of the etiology ,109,110.Some studies showed that during the proliferative phase of the menstrual cycle β-catenin is mainly localized in the nucleus whereas during the secretory phase β-catenin translocated from the nucleus to the cytoplasm and cell membrane . EstradiInformation on the importance of Wnt ligands in endometrial adenocarcinoma is increasing and it is likely that Wnt alteration plays a role in carcinogenesis. Bui et al. documented that Wnt4 mRNA is higher in normal endometrium than endometrial carcinoma; WNT2, WNT3, and WNT5A mRNA levels are similarly higher in normal endometrium than in endometrial carcinoma. This study suggests that down-regulation of WNT4, WNT2, WNT3, and WNT5A might be important in the development of endometrial cancer ,114. HowWNT7 gene expression. The expression of WNT7A and WNT7B genes is high in endometrial carcinoma cell lines but not in normal primary endometrial cultures [WNT7A and WNT7B can be detected in normal human endometrial tissues and human endometrial tumors. This discrepancy is likely due to the artificial environment and regulatory signals that control WNT7A and WNT7B expression [WNT7A gene was absent or reduced and was negatively linked with FIGO stage, grade, lymph node metastasis, depth of myometrial invasion, and peritoneal cytology in approximately 60% of patients [WNT7A gene was overexpressed in most cases of endometrial cancer [WNT7A positively correlated with overall survival and disease-free survival in endometrial cancer patients [WNT7A plays a pathogenic role.Much of the research to date has focused on cultures ,116,117.pression . In one patients , suggestWNT7A, the expression of WNT10A and WNT10B ligands has been associated with estrogen-related carcinogenesis of endometrial cancer. Wnt10b protein expression is significantly higher in endometrial cancer tissue in comparison to hyperplastic endometrium and normal endometrium [WNT10A gene in endometrial cancer, aside from its upregulated expression, remains unknown [In addition to ometrium . Moreoveometrium ,119. How unknown .PTEN function is linked with endometrial cancer onset and PTEN is frequently mutated in endometrioid endometrial tumors [PTEN function loss stimulates defects in the function of the APC that normally regulate the formation of the β-catenin “destruction complex” that in turn causes constitutive activation of the Wnt/β-catenin pathway and promotes the development of cancer. The result of the constitutively activated Wnt/β-catenin pathway is squamous cell metaplasia (SCM) with no malignant transformation. Consequently, it appears that activation of the Wnt/β-catenin pathway accelerates rather than initiates the cancer while the simultaneous loss of PTEN activity and activation of the Wnt/β-catenin pathway is associated with the development of an aggressive form of endometrial cancer [Studies by van der Zee et al. demonstrated a synergistic effect of the Wnt/β-catenin and PTEN pathways in endometrial cancer . Loss ofl tumors . PTEN ful cancer .CTNNB1). Between 10% and 45% of endometrial cancers have missense mutations of CTNNB1 [CTNNB1 mutations are apparently found in the early stages of endometrial carcinogenesis [CTNNB1 mutation is primarily detected in endometrioid endometrial cancer rather than nonendometrioid endometrial carcinoma cases (NEEC) [The other major alteration reported in endometrial cancers is nuclear accumulation of β-catenin  ,122,123.CTNNB1 mutation frequency in 29 endometrial cancer tumors with or without microsatellite instability (33% MSI-H and 50% MSS/MSI-L), suggesting that there is no correlation with the presence or absence of underlying microsatellite instability [CTNNB1 mutation frequency in endometrioid endometrial cancer [CTNNB1 mutations and concomitant nuclear expression of β-catenin were found in 16.8% of the 125 endometrial carcinomas studied. Nuclear localization of β-catenin in neoplastic cells varies between 5% and 60% in CTNNB1 mutant endometrial carcinomas (mean 19.8%) [CTNNB1 mutations were found in 63 (18%) of 345 endometrial cancer patients; 53 of these patients had tissue available for immunohistochemistry. Among these 53 samples, 46 were of pure endometrioid histology and seven were mixed endometrioid and nonendometrioid carcinomas. This study found that 45/53 (85%) of CTNNB1 mutant endometrial cancers had nuclear β-catenin localization [CTNNB1-mutant endometrial cancers as other cellular components may also help activate the Wnt pathway. Detailed CTNNB1 mutational rates are listed in β-catenin mutations in exon 3, on the serine/threonine residue that is the site of regulatory phosphorylation by GSK3β, have been identified ,124. Muttability . Schlosstability ,91, whill cancer . Recentln 19.8%) . Anothern 19.8%) . Kim et lization . Taken tAnother study by Shelton and Goodheart et al. examined the role of lymphoid enhancer-binding factor 1 (Lef1) in endometrial cancer . Lef1, aPrecision medicine is often limited by the identification of biological mediators, the pathology of various conditions, and the development of treatments that efficiently target specific genes; in addition, treatments often have side effects. Recent research has significantly increased our understanding of the function of Wnt secretion in carcinogenesis and revealed novel therapeutic targets. The Wnt cascade constitutes potential targets for pharmacological intervention and use in personalized medicine; however, the role of the individual β-catenin pathway components must be better elucidated in endometrial cancer. Currently, very few effective treatment strategies exist for advanced endometrial cancer patients who have failed traditional chemotherapy. Novel biologics targeting VEGF and mTOR pathways have shown promising results for endometrial cancer in phase II clinical trials ,137. CTNCurrently, numerous β-catenin-dependent signaling inhibitors are available as pre-clinical, trial, or FDA-approved therapies, including MPA, DKN-1, OMP-54F28, Niclosamide, and PRI-724 among others.Wang et al., in a study of 21 patients, showed that a synthetic form of progesterone  counteracts the proliferative effects of E2 (estradiol) during the normal menstrual cycle, hyperplasia, and early endometrial carcinogenesis by inhibiting Wnt/β-catenin signaling gene expression . While l2. Of 47 evaluable patients, the 12-month response rate was 83%, 37 were complete responders , 2 were partial responders , 3 had stable diseases , and 5 had progressive diseases . Westin has also reported on genes that are important in cell proliferation, estrogen signaling, and Wnt signaling [In a clinical study, Westin and his research team  documented the effectiveness of a levonorgestrel-releasing intrauterine system in treating patients with complex atypical hyperplasia or grade I endometrial cancer . The aimignaling .DKN-01 is a humanized monoclonal antibody (Mab) targeting Dickkopf-1 (DKK1) and is being developed as an antineoplastic agent. One study (NCT03395080), started in Feb 2018 and funded by Leap Therapeutics, Inc., uses DKN-01 as a monotherapy or in combination with paclitaxel in patients with recurrent epithelial endometrial or epithelial ovarian cancer or carcinosarcoma . Doses oWnt-driven cancers can be targeted at many points in the pathway ,57,138. OMP-54F28 is a fusion protein consisting of the extracellular ligand-binding domain of Fzd8 and a human immunoglobulin G1 (IgG1) Fc domain. This decoy receptor interferes with Wnt signaling by sequestering secreted Wnts and shows antitumor activity in several patient-derived xenograft (PDX) models ,150. CurNiclosamide (trade name Niclocide), an FDA-approved salicylamide derivative used for the treatment of tapeworm infections, targets the Wnt/β-catenin pathway. Recently, it was observed that niclosamide inhibits the β-catenin-dependent pathway in ovarian tumor cells isolated from 34 patient ascites . MoreoveBesides the intracellular perturbation of Wnt secretion and ligands, an inhibitor of the downstream Wnt pathway is currently undergoing clinical trials. Emami et al. developed a small molecule inhibitor (PRI-724) closely related compound ICG-001 that targets the complex formation of β-catenin and CBP that specifically downregulates the expression of β-catenin-TCF-responsive genes . TreatmeSalinomycin, an antibiotic potassium ionophore, is a selective inhibitor of breast cancer stem cells . SalinomCurcumin inhibits the initiation, promotion, and progression of carcinogenesis. Curcumin may exert anticancer effects through a variety of pathways that are involved in mutagenesis, apoptosis, tumorigenesis, cell cycle regulation, and metastasis. The anticancer effects of curcumin involve in the activation of apoptotic pathways as well as in inhibition of tumor microenvironments . Extensive studies have demonstrated that curcumin targets several therapeutically relevant cancer signaling pathways, such as p53, Ras, PI3K, AKT, Wnt/β-catenin, and mTOR . ClinicaMicroRNAs (miRNAs) are small noncoding RNAs comprised of about 22 nucleotides that are a class of naturally occurring small noncoding RNA molecules that regulate cellular function by controlling multiple target messenger RNAs. A single miRNA may target at least 200 genes and a single gene can be regulated by many RNAs. Since individual miRNAs have potentially numerous target genes, miRNA dysregulation can profoundly regulate the cellular machinery and facilitate cancer hallmarks . NumerouCTNNB1 to suppress Wnt/β-catenin signaling in SGC790 and U251 cells [Wang et al. documented that miR-15a-5p levels are significantly decreased in endometrial cancer compared with healthy controls both in in vitro and in vivo . In this51 cells . Moreove51 cells ,181. ThuTo date, the use of various inhibitors of Wnt/β-catenin targeting cancer as prognostic markers and tumor suppressors has been reported and novel agents targeting the Wnt/β-catenin signaling pathway have shown encouraging results. Endometrial cancer should be the focus of these initiatives with the goal of establishing Wnt pathway components as prognostic and predictive biomarkers as well as demonstrating preclinical evidence and assessing the efficacy of established inhibitors for this pathway.The Wnt signaling pathway has been shown to be involved in crosstalk with several other signaling pathways, such as the Notch, mTOR, Hedgehog, and estrogen signaling pathways, and with the tissue microenvironment. Since endometrial cancer pathogenesis involves various genomic changes and aberrant signaling pathways; investigating the association between the incidence of these molecular disorders and their plausible crosstalk will potentially reveal important targets for synergistic drug combinations in endometrial cancer and advance the development of targeted and more successful therapies ,183.Cancer stem cell (CSC) activation of the Wnt pathway has been shown to initiate a complex transcriptional program resulting in the expression of genes involved in drug resistance (such as ABCB1 and CD44) and immune function (such as PD-L1 and CD47) . A recenWnt signaling acts as a bridge between tumor cells and the tumor microenvironment contributing to cancer progression, drug resistance, and immunity escape . While WIncreasing evidence for the crosstalk between Wnt/β-catenin and autophagy suggests the interplay between both pathways as an attractive therapeutic target . HydroxyUnopposed estrogen and Wnt signaling aberrations are major pathways accounting for the endometrial cancers. A study using a uterine epithelial cell specific inducible Cre mouse model and 3D in vitro culture of human endometrial cancer cell lines demonstrated interplay between hormone and Wnt signaling pathways in endometrial cancer. This study showed that activating mutations in the Wnt signaling pathway for a prolonged period is insufficient to cause endometrial cancer but leads to development of endometrial hyperplasia. However, together with unopposed estrogen, activating mutations in Wnt signaling drive the progression of endometrial hyperplasia to endometrial cancer. Overall, this study indicated use of progesterone as a targeted therapy for endometrial cancer patients with an activated Wnt signaling pathway . AnotherAbnormal activation of WNT signaling has been reported in the majority of type-1 endometrial cancer cases with β-catenin mutations in 20–25% of cases. Given the current lack of treatment options for advanced and recurrent endometrial cancer patients and the growing body of evidence supporting the role of Wnt signaling at early stages of endometrial carcinogenesis, Wnt signaling represents a promising intervention for targeted therapies in endometrial cancer patients. Various inhibitors targeting different molecules of this pathway have been developed, though only a few studies have addressed the effects of Wnt inhibitors in endometrial cancer and they are still at an early phase and far away from clinical trials. Future studies evaluating the efficacy and safety of Wnt/β-catenin signaling inhibitors in endometrial cancer are needed to aid in the development of targeted novel therapies that can reverse the increasing trend of mortality in endometrial cancer patients. With the recent advances in genome editing, further research utilizing human-derived organoid models and tissue-specific inhibition of Wnt signaling will enhance our understanding and identify novel targets of the Wnt/β-catenin signaling pathway in endometrial cancer. Since combination therapy with other cancer drugs has been shown to limit off-target toxicities and considering the contribution of the crosstalk between Wnt signaling and the host immune response to therapeutic resistance and immune escape, Wnt inhibitors in combination with immunotherapies must be evaluated in endometrial cancer to improve both overall treatment efficacy and patient outcomes.


In the literature on inter-limb coordination, the coordination among ‘‘focal’’ body parts  directly engaged in a pendulum swinging task has been studied by immobilizing other body parts to reduce “noise,” while putting aside questions of how one maintains posture while performing the task. However, in practical performance of musical instruments, for example, performers must coordinate different body parts in sync with the music while maintaining the whole body’s balance. This study demonstrates the effectiveness and necessity of understanding inter-limb coordination in whole-body coordination. Participants were asked to move two pendulums either in sync or alternatively with metronome beeps under two conditions: immobile (fixed forearms) and mobile (forearms not fixed). The explorative analyses focused on whether and how coordinative structures emerged and whether the degree of task achievement differed according to the phase mode, frequency, and mobility conditions. The motion similarity and phase difference between different parts and the pendulums showed that task-specific coordinative structures emerged in both immobile and mobile conditions. In the in-phase mobile condition, the emergent coordinative structure may have improved task achievement, shown by the phase difference between the left and right pendulums. These findings suggest that the global coordinative structure is involved in achieving the local pendulum swinging task. In the performance of a musical instrument, dance, etc., performers are required to coordinate different body parts in sync with the rhythm of the music while simultaneously controlling posture to maintain the balance of the whole body. For example, in drum playing, wherein one beats the instruments by using sticks in both hands and kicking the foot pedals, a skilled drummer employs the whole body, including the head and trunk. Such control in musical performance has been studied while concentrating on “focal” inter-limb coordination in the framework of the dynamical systems approach (DSA).DSA considers the body as an open non-equilibrium system and provides a mathematical model to describe body movements as a self-organizing phenomenon affected by intrinsic and extrinsic constraints. Studies by Kelso et al. showed that control parameters such as frequency affect the behavior of periodic inter-limb coordination synced with metronome beeps . For exaTo explain and predict human periodic motion in dynamic terms, Haken, Kelso, and Bunz  proposedAlthough previous studies have successfully elucidated the inter-limb coordination in terms of the HKB model by limiting their observation to fully controlled experiments, they have not addressed more realistic situations  in which performers are required to achieve tasks such as postural control, as mentioned at the beginning of this paper. It is thus essential to understand the local coordination movements between body limbs  as not independent from the other body parts but related to postural control of the whole body, including elbows, shoulders, trunk, and head.Based on the idea of Bernstein , motor cTo fully understand more realistic movements such as playing a musical instrument or dancing, it is necessary to examine whether and how the global coordinative structure in relation to the focal body movements emerges to maintain the posture while allowing one to achieve the task when the body is unconstrained. The present study aims to explore and describe this.Thus, this exploratory study aims to identify global coordinative structures that emerge in the pendulum-swinging task using the left and right limbs  and to specify the relationship between the global structure and local task achievement. To achieve this goal, while keeping factors such as relative phase modes (in-phase and anti-phase) and task frequencies (six levels) more or less the same as in previous studies, we conduct an experiment to see the effects of the “mobility” factor, which consists of the following two conditions: (1) the “immobile” condition, wherein the participants’ forearms are fixed to the tabletop and their mechanical interaction with the other body parts are not allowed, (2) and the “mobile” condition, wherein the forearms are unconstrained and their mechanical interaction with the other body parts is allowed. All tasks are performed while the participants are seated on a stool.The data are analyzed from two perspectives: whether and how the global coordinative structure emerged and the degree of task achievement. Regarding the global coordinative structure, the motion similarity  and phase difference  of the pendulum to each body part  are calculated and compared among conditions. Regarding the degree of task achievement, the mean and standard deviation (SD) of the phase difference between the left and right pendulums are calculated and compared among the conditions. We analyze the motion data of the anterior-posterior axis, where the periodicity of the pendulum is more clearly shown than that of the other axes.We consider that the mobile condition enables more flexibility for the human-pendula system to reorganize different demands in different conditions in the task frequency and relative phase mode. Accordingly, in the mobile condition, compared with the immobile condition, we predict that movement is more organized in a way such that the moving hands and/or pendulums are not only compensated for by but are also embedded in the global coordinative structure, including forearms, upper arms, trunk, and head. Additionally, we predict that the global coordinative structure varies depending on the task frequency and relative phase mode, and the degree of task achievement is higher in mobile conditions than in immobile conditions.SD = 1.27 years; mean height = 171.43 cm, SD = 7.36 cm). They were all recruited from the School of Human Sciences or Graduate School of Human Sciences, Waseda University.Eighteen students from Waseda University participated in this experiment. They were all male and right-handed students, without any injuries or other health problems to participate this experiment , metal weight (191 g), and bicycle handlebar grip . When the pendulums were held by a participant, a medical supporter was wound tightly around the holding hand to avoid creating a space between the pendulum and the palm.A desk with a tabletop, whose height was adjustable between 72 and 123 cm was used as an armrest .A stool, whose height could be adjusted, was used to allow the participant to be seated .A metronome was used to maintain the tempo of the experimental task .A pair of headphones was used for the participant to listen to the metronome sound .A three-dimensional opto-electronic motion-capture system  with 12 cameras (camera version: Flex 3) was used to record at a sampling rate of 100 Hz the movements of the pendulum and the participant’s body parts (see the section on procedure for the details) where the reflective markers were attached.A data acquisition system  was used to synchronize and record motion and metronome data that underwent analog to digital conversion (100 Hz).The participants performed a pendulum-swinging task using both hands. They were instructed to move a pair of pendulums each held by their left and right hands, to the beat of the metronome beeps. The experimental design had a 2 × 6 × 2 within-participant design; the first factor was the mobility of the forearms (mobile vs. immobile); the second factor was the frequency of swinging movements ; and the third factor was the relative phase (in-phase and anti-phase). In each trial, the measurement was started after the swinging movements reached 10 cycles, and the measurement lasted till we obtained 30 movement cycles for each trial .To avoid unnecessary variances in the data, and to counterbalance the effect of order by fastening or loosening the Velcro tapes in every trial (detailed below), nine participants performed the mobile condition first and the immobile condition later, while the remaining nine participants performed the tasks in the reverse order. The order of the other 12 conditions was randomized for each block.On arrival, the participants were informed of the procedures of the experiment and signed an informed consent form on their own will. The participants then changed into a motion capture suit and had a cap on  with reflective markers on the vertex, C7, both acromia, elbow joints (olecrana of both ulnae), and wrist joints (heads of both ulnae). Meanwhile, the height of the eyes in the upright seated position was marked on the wall, and the participants were instructed to keep their eyes on the marker as they performed the task. The height of the tabletop was adjusted such that the forearms were horizontal. During each trial, the participants were instructed to synchronize the movements of the pendulum to the presented metronome beeps as accurately as possible. In the in-phase condition, the tips of the pendulums came to the point closest to the body as the metronome beep sounded . In the anti-phase condition, the tip of the right-hand-held pendulum came to the point closest to the body, and the tip of the left-hand-held pendulum came to the point farthest from the body as the metronome beep sounded . The participants were further instructed to move the pendulum as straight, continuously, and smoothly as possible under all conditions. The participants practiced until they could perform the task under all conditions, as instructed. In the immobile condition, the forearms were fastened with two Velcro tapes tightly to the armrests and were adjusted to be horizontal . In the This study was reviewed and approved by the Ethics Review Committee on Human Research of Waseda University (2017–140) and was performed in accordance with the Declaration of Helsinki.In the data analysis, we used the time series of the anterior-posterior motion of the body and the pendulums. The first 10 cycles were discarded to remove the transient fluctuations in the data. The remaining data were bandpass-filtered using a 0.1–10 Hz Butterworth filter  and z-scored.Because we wanted to know whether and the extent to which the left and right pendulums and body parts are related to each other, the first peak value of cross-correlations (as a measure of similarity) and relative phases (as a measure of phase difference) of all of these in relation to the right and left pendulums were calculated. The lag in calculation of the cross-correlations was set to one cycle before and after. The relative phases were obtained as follows. The motion data underwent the Hilbert transformation to obtain the phase angles. Then, the continuous relative phases of each body part relative to the left and right pendulums were calculated. Because we only focused on the phase difference in this analysis, we calculated the average of the relative phases. Therefore, 18 relative phases and 18 cross-correlation coefficients were obtained .To visualize the common coordination among participants, the above two indices were averaged and presented as lines projected onto the topologically arranged body parts see Figs  and 3. Tϕ, respectively. Continuous relative phases (ϕ) of the left and right pendulums were computed using the same method as in the previous section (ϕ = θright − θleft). The phase shift and the absolute value of the phase shift were calculated using circular statistics [ϕ) and the prescribed relative phase mode . Additionally, the absolute value of the phase shift was obtained. The phase shift and its absolute value were meant to indicate the extent to which participants achieved the task. Meanwhile, SDϕ, calculated using circular statistics [The degree of task achievement and phase stability of the left-right pendulums were evaluated based on the phase shift and SDatistics , 16; theatistics , 16, repϕ, and relative phase between body parts), the Harrison-Kanji test was employed, and the two-way ANOVA was applied to the Fisher z-transformed cross-correlation coefficients. The Harrison-Kanji test is used for the ANOVA of angular data, allowing the examination of two-way main effects and interaction [To examine the main effect and interaction of the two factors on each index , while there were unignorably large individual differences for phase differences between C7/head and the pendulum, a characteristic pattern of phase differences between the shoulder and the pendulum was observed in both the mobile and immobile conditions .In the immobile condition, at low frequencies (1.5 Hz and 1.7 Hz), phase differences between the shoulder and the pendulum of the same side were close to 0 degree (red), and those of the opposite side were close to 180 degrees (blue). However, as the frequency increased, phase differences between the shoulder and the pendulum, regardless of sides, became approximately 90 degrees (light green), similar to the patterns observed for the wrists and elbows.In contrast, in the mobile condition, phase differences between the shoulder and the pendulum of the same side  were closer to 180 degrees (blue), and those of the opposite side  come close to 0 degree (red) as the frequency increased.A two-way analysis of variance (ANOVA) on the first peak values of the cross-correlation showed that, in the in-phase condition, the main effect of the mobility factor was observed in all 18 combinations, and the correlation values in all combinations, except for R-pendulum and L-pendulum, were higher in the immobile condition than in the mobile condition , the frequency’s main effect was significant in both the immobile and mobile conditions, and the main effect of mobility factors was observed in each frequency. In other words, for the mobile and immobile conditions, the phase difference between the pendulums and the remote body parts varied with frequency, and the pattern of variation was qualitatively different in the mobility factor.In the anti-phase condition, interactions were observed between mobility and frequency for body parts in the immediate vicinity of the pendulum . This corresponds to the counterclockwise transitions in the phase differences between the pendulum and the body parts in the vicinity of the pendulum  as the frequency increases. This can be interpreted as an effect of the mechanical connections among the body parts. Furthermore, in the anti-phase condition, unlike in the in-phase condition, no main effect or interaction was observed for the head or C7. In other words, we cannot say that the phase difference between the head and C7 changed depending on the frequency or mobility factors.F = 4.527, p=0.035; Frequency: F = 2.527, p=0.03. Post-hoc tests revealed that the absolute value of the phase shift was significantly smaller in the mobile condition than in the immobile condition, and no significant difference was confirmed for frequency in any pair (adjusted p>.05). These results show that the participants were better in an attempt to maintain the in-phase mode in the mobile condition than in the immobile condition.Results of the two-way Harrison-Kanji test conducted on the phase shift and absolute phase shift for mobility and frequency in the in-phase mode condition are shown in ϕ  = 9.586, p=0.002; Frequency: F = 13.227, p<.001. Post-hoc tests showed that SDϕ was significantly smaller in the mobile condition than in the immobile condition. It also showed that SDϕ was significantly different in the following comparisons: 1.5, 1.7 Hz < 1.9, 2.1, 2.3, 2.5 Hz, and 1.9 Hz < 2.5 Hz (adjusted p<.05). These results imply that while the task performance in keeping the in-phase mode became increasingly unstable as the frequency increased, it was achieved more stably in the mobile condition than in the immobile condition.For the SDϕ , while tF = 3.912, p=0.002. The post-hoc test showed that the absolute value of phase shift was significantly smaller in the 1.5 Hz condition than in the 2.3 Hz condition (adjusted p<.05). Therefore, the degree of deviation from the targeted phase mode, that is, anti-phase, increased with frequency. For the SDϕ  = 5.209, p<.001. The post-hoc test showed that SDϕ was significantly larger in the 2.5 Hz condition than in the 1.5 Hz and 1.7 Hz conditions (adjusted p<.05). Collectively, as in the in-phase condition, the phase difference became more unstable as the frequency increased, but it did not change regardless of mobility.Results of the two-way Harrison-Kanji test for mobility and frequency for the anti-phase mode are shown in  the SDϕ , there wHere, we put the results into perspective and discuss whether and how global coordinative structures emerged under the conditions set in this study. Let us start by confirming the constraints in different conditions set for the present experiment.Now, in the immobile condition, in comparison with the mobile condition, the following were found: regardless of the phase mode, all of the body parts, except for shoulders in the anti-phase condition, exhibited a higher degree of motion similarity with the right pendulum Figs  and 2, aIn the mobile condition, there are only three surfaces of contact with the environment: the buttocks and a pair of soles; thus, the whole body is less stable than in the immobile condition with five surfaces of support: the buttocks, a pair of soles and the forearms. Accordingly, a more compensatory motion is required to perform the pendulum task in the mobile condition than in the immobile condition. However, because there were more degrees of freedom in the mobile condition, it is possible that the participants were able to use them to regulate the whole body to achieve the pendulum task while maintaining the posture. The way in which the participants used the degrees of freedom seems to depend on the phase mode, and different patterns seem to have emerged.Given the above consideration, the higher degree of similarity in motion between the body parts and the pendulums in the immobile condition can be interpreted as an emerging coordinative structure in which the impact of the pendulums is dispersed and absorbed by the five surfaces of support. Phase differences did not change significantly regardless of frequency, because the whole body was more stable with more support surfaces in this condition than in the immobile condition. That is, the demand to compensate for the movement of the pendulum weight to maintain the sitting position may have been much less in the immobile condition than in the mobile condition.Note that these findings of higher similarity in motion between the pendulums and most of the body parts were obtained under the condition wherein the forearms were tightly fastened to the tabletop. That is, the table and the forearms as a whole might have moved more or less together by swinging the pendulums.Meanwhile, in the mobile condition, we observed characteristic coordination patterns for both the frequency and phase modes.As for the in-phase condition in the mobile condition, the remote body parts (shoulder and C7) and the pendulums were in anti-phase  to 20 degrees 2.5 Hz; . This suAs for the anti-phase mobile condition, the following were observed: The shoulders, but not others, showed higher similarity in motion to the pendulum compared with the immobile condition . FrequenIn the anti-phase condition, the movement of two pendulums in opposite directions at a distance of the shoulder width in effect rotates the torso, as shown in Taken together, when the body was unconstrained to move, a global coordinative structure emerged according to the demands of the in-phase and anti-phase conditions, respectively. Let us now discuss the global coordinative structure in relation to the task achievement.ϕ, phase shift, etc.) and the depth using SDϕ, etc [ϕ is larger, the basin of attraction can be estimated to be shallower.As described by the HKB model , the posSDϕ, etc , 3, 20. ϕ and the phase shift were smaller in the in-phase mobile condition than the immobile condition, we can infer in dynamical terms that there was an attractor common to the participants, and that it was deeper as well as closer to the prescribed relative phase in the in-phase mobile condition than in the in-phase immobile condition.Given that both SDϕ was slightly larger than that of the in-phase condition as ϕ. This suggests that the attractor depth was equivalent in both mobility conditions. Additionally, given that the inter-participant variability was larger in the immobile condition than in the mobile condition, the position of an attractor between participants may well have been more diverse in the immobile condition than in the mobile condition, but at the equivalent depth. Thus, in light of the HKB dynamics, in the anti-phase immobile condition it is possible that there was no common strong attractor to all participants, with instead a different attractor for different individuals, in contrast to the anti-phase mobile condition. In such a case, the underlying dynamics may possibly be different for the two conditions.As for the anti-phase condition, while SDIn the present study, we obtained findings suggestive of extending the conventional model of inter-limb coordination  to more practical motor tasks. That is, in the mobile condition, while the focal phase difference (in-phase or anti-phase) was maintained , the phase difference between the torso and the pendulum exhibited an anti-phase pattern.Although the anti-phase has been characterized as a less stable state in the conventional two-oscillator model, it may be considered an important state that supports focal stability in the background when viewed from the perspective of the whole-body coordinative structure, including posture.The idea of “beyond HKB” may not be very new , 21, 22,Finally, we discuss the implications of the research on musical performance. In this study, we confirmed that the global coordinative structure varies with the task-dependent states of the local body parts .As alluded to earlier, in musical instrument performance, motion of the local body parts in direct contact with the instrument can be considered as essentially inseparable from the postural control of the whole body. Then, how are the local body parts directly involved in producing sound coordinated with the maintenance of posture? How is coordination regulated by the characteristics of the instrument and the composition of music? Do these depend on the skill level?Attempts have been made to answer some of these questions. For example, a study on drum-set playing  showed tSimilarly, in research on violin  and cellSome coordinative structures specific to instrumental playing can be only revealed by looking at local body parts. To truly understand the mechanism of instrumental performance, it is essential to explore the instrument/expert-specific structures that emerge in the global coordinative structure in relation to the local coordinative structure.


The apparent activation energy, aE, has been calculated by several differential and integral isoconversional methods, namely Kissinger, Friedman, Flynn–Wall–Ozawa (FWO), Kissinger–Akahira–Sunose (KAS) and Starink. All methods provide similar values of aE (between 66 and 69 kJ/mol), and this shows independence versus the heating rate used. The epoxy resins crosslinking is characterized by a multi-step process. However, for the sake of the simplicity and to facilitate the understanding of the influence of the oxirane location on the curing kinetic, this can be assimilated to a single-step process. The reaction model has a high proportion of autocatalytic process, fulfilling that Mα is between 0 and pα and Mα < n and m) it is possible to obtain, on the one hand, the kinetic parameters and, on the other hand, a graphical comparison of the degree of conversion, α, versus temperature (T) at different heating rates with the average n and m values of this model. The good accuracy of the proposed model with regard to the actual values obtained by DSC gives consistency to the obtained parameters, thus suggesting the crosslinking of the ELO-based epoxy has apparent activation energies similar to other petroleum-derived epoxy resins. The curing process of epoxy resin based on epoxidized linseed oil (ELO) is studied using dynamic differential scanning calorimetry (DSC) in order to determine the kinetic triplet ( Flax is one of the industrial crops that, thanks to its versatility, has been used since ancient times both in food and in clothes manufacturing for approximately 30,000 years . NowadayOne of the most interesting by-product is flax oil (or linseed oil), which represents approximately 45% of the seeds ,7. The mBy taking into account the high degree of unsaturated carbons contained in the main fatty acids, which represents about 73%, its chemical modifications opens a broad potential for industrial applications ,12. SomeAs above-mentioned, one of the most common chemical modification on unsaturated fatty acids is epoxidation. This increases the reactivity of the linseed oil since the oxirane rings are much more reactive than unmodified unsaturations in fatty acids ,17. EpoxaE, the reaction model, A, are determined. The main aim of this work is to compare the kinetic triplet of the ELO-based epoxy resin with other petroleum-derived epoxies to assess the effect of non-terminal oxirane groups in ELO with regard to conventional epoxy resins with epoxy groups located in terminal and more readily available positions. Furthermore, through the comparison of the experimental functions y(α) and z(α), with standard master plots and the determination of the m and n parameters of Šesták–Berggren model ), the influence of autocatalysis process and the accuracy of the kinetic triplet parameters are determined.This crosslinking process between an epoxy resin and the hardener provides an exothermic reaction, with an important release of heat that can contribute to increase the temperature, and subsequently, the possibility to provide an autocatalytic process. In previous works, the curing kinetics of a bisphenol A-based resin with a content of 31% by weight of ELO was studied, and it was observed that the mechanical properties obtained were similar to those of a conventional petroleum-derived epoxy resin . In the 3 at 20 °C. The epoxy equivalent weight (EEW) of the ELO-based epoxy resin was 178 g/equiv. A liquid at room temperature cyclic anhydride, namely, methyl nadic anhydride (MNA), with an anhydride equivalent weight (AEW) of 178.18 g/equiv was employed. This anhydride-based hardener was employed due to epoxy/anhydride systems have higher glass transition and less shrinkage, interesting properties if the objective is the use of ELO resin in industrial applications  and independently of the conversion rate α.The curing kinetics of bio-based epoxy resin from epoxidized linseed oil has been broadly analyzed using dynamic DSC at four different heating rates . The apparent activation energy (y(α) and z(α) versus conversion, α, allowed obtaining the parameters denoted Mα and Mα is between 0 and pα and 2: Mα < Mα (0.1038), indicates that the autocatalytic effect is greater at low heating rates (10 °C/min). The reaction model, A, were obtained through the Šesták–Berggren parameters ). Theoretical curves plotted applying the values of kinetic triplet obtained show a great concordance with the experimental data, confirming the accuracy of the parameters obtained for an epoxy resin based on ELO. This widens the use of flax crops as linseed oil can be converted into a biobased epoxy resin with similar curing/crosslinking parameters to those of petroleum-derived epoxy resins.The analysis of the highest point in the graphical representation of parameters


BMAL1 knockdown (KD) and Wnt/β-catenin pathway inhibition were performed in chondrocytes. The contents of proinflammatory factors and OA biomarkers in serum and chondrocyte secretions were detected by ELISA. Pathological and immunohistochemical staining of articular cartilage indicated the deterioration of cartilage. WB and qPCR were used to evaluate the relationship between matrix degradation and the activation of Wnt/β-catenin signaling pathway in chondrocytes. We found that chronic CRD could cause OA-like pathological changes in knee cartilage of rats, accelerating cartilage matrix degradation and synovial inflammation. The expression of MMP-3, MMP-13, ADAMTS-4, and β-catenin increased significantly; BMAL1, Aggrecan, and COL2A1 decreased significantly in either LD-shifted cartilage or BMAL1-KD chondrocytes. The expression of β-catenin and p-GSK-3β elevated, while p-β-catenin and GSK-3β diminished. The inhibitor XAV-939 was able to mitigated the increased inflammation produced by transfected siBMAL1. Our study demonstrates that chronic CRD disrupts the balance of matrix synthesis and catabolic metabolism in cartilage and chondrocytes, and it is related to the activation of the canonical Wnt/β-catenin signaling pathway.With the gradual deepening of understanding of systemic health and quality of life, the factors affecting osteoarthritis (OA) are not limited to mechanical injury, metabolic abnormality, age and obesity, etc., but circadian rhythm, which plays a non-negligible role in human daily life. The purpose of this study was to explore the molecular mechanism of chronic circadian rhythm disturbance (CRD) inducing cartilage OA-like degeneration. Rats with the anterior cruciate ligament excision transection (ACLT) were used to establish the early-stage OA model (6-week). The light/dark (LD) cycle shifted 12 h per week for 22 weeks in order to establish a chronic CRD model.  Environmental factors can significantly affect body metabolism, especially light, temperature and humidity. Light/dark (LD) cycle is the most important environmental zeitgeber that affects circadian homeostasis. Irregular LD cycles can disrupt the body’s circadian rhythm, even metabolic disorders, especially in shift workers . PreviouBMAL1−/− mouse joints. Additionally, BMAL1 can regulate the activity of chondrocytes through HIF1α-VEGF and TGF-β signaling pathways . However, these illustrate an interesting phenomenon that these theories are contrary to the previously proposed expression of BMAL1 and the Wnt pathway in OA cartilage. Therefore, can BMAL1 still cooperates with Wnt/β-catenin pathway in OA? Or whether it’s relevant? In this study, we established chronic CRD models in rats and chondrocytes, in order to study the effects of CRD on rat articular cartilage and the relation between clock proteins and the Wnt/β-catenin pathway.Numerous studies have shown that clock genes are involved in activation and inhibition of the Wnt/β-catenin pathway . KnockdoAntibodies used for western blotting (WB) and immunostaining included mouse anti- COL2A1 , rabbit anti-β-catenin , rabbit anti-BMAL1 , rabbit anti-MMP-3 , rabbit anti-MMP-13 , rabbit anti-ADAMTS-4 , rabbit anti-Cyclooxygenase-2 (COX-2) , rabbit anti-β-catenin , rabbit anti-GSK-3β , mouse anti-p-β-catenin , mouse anti-p-GSK-3β , mouse anti-GAPDH , Alexa Fluor® 488-conjugated goat anti-mouse immunoglobulin G (IgG) (H + L) , Cy3 conjugated goat anti-rabbit IgG (H + L) , Horseradish Peroxidase (HRP) goat anti-mouse IgG (H + L) , and HRP goat anti-rabbit IgG (H + L) .siRNA medium mixture (serum-free medium + siBMAL1 (20 μM))  were prepared and transfected. Sequences for negative control and siBMAL1s are listed in Primary chondrocytes were isolated from tibial platform of rats under sterile conditions, released by treating with 0.25% Trypsin-EDTA and 0.2% Type II collagenase  at 37°C (5% CO2) in Dulbecco’s Modified Eagle’s Medium (DMEM)/Hams F-12, supplemented with 10% fetal bovine serum (FBS)  and 1% Penicillin/Streptomycin. After passage, a second generation of chondrocytes was used for the experiment. For cell transfection, chondrocytes in logarithmic growth phase were taken and transfected when the density reached 60–80%. GP-Transfect-Mate medium mixture (serum-free medium + GP-Transfect-Mate) and 4 cells/ml, and cultured in 6-well plates. Cells were fixed with 4% paraformaldehyde, permeabilized, and then blocked with 3% bovine serum albumin (BSA) at room temperature for 30 min. After removing the blocking solution, chondrocytes were incubated with primary antibody overnight at 4°C. COL2A1 (1:100) and DAPI for chondrocyte identification, β-catenin (1:100) for nuclear translocation. The cell climbing slides were washed and covered with fluorescently labeled secondary antibody for 50 min at room temperature. DAPI was incubated for 10 min at dark. Fluorescence was observed under a fluorescence microscope  at 545 nm (red) and 340 nm (blue).The secondary generation chondrocytes were diluted to 2×102O  were subjected to 40 amplification cycles. Primer sequences are listed in ®480  to obtain the cycle threshold (Ct).Ribonucleic acid (RNA) in the chondrocytes was extracted at 4°C using SV total RNA isolation system , the concentration was measured at 260 and 280 nm, and then reverse transcribed blending with GoScript™ Reverse Transcription Mix, Oligo (dT)  into cDNA (1 μg/ml). The mixture of 10 μl GoTaq®qPCR Master Mix, 0.6 μl forward and reverse primers, 2.5 μl cDNA and 6.3 μl RNase-free ddHTotal protein samples of chondrocytes were extracted following the instructions . The mixture of radio immunoprecipitation assay (RIPA) and Phenylmethylsulfonyl fluoride (PMSF) fully lysed the cells. Then equal 20 μg amounts of protein were separated onto 10% SDS-PAGE and transferred to nitrocellulose (NC) filter membrane. The membranes were blocked in 5% nonfat milk dissolved in Tris Buffered Saline with Tween®20 (TBST) buffer and probed with primary antibodies for BMAL1 , MMP-3 , MMP-13 , ADAMTS-4 , COX-2 , β-catenin , GSK-3β , p-β-catenin , p-GSK-3β , and GAPDH  at 4°C overnight followed by secondary antibody coupled with horseradish peroxidase at room temperature for 2 h. The blots were developed using the ECL system  and the gray value was quantified with Image J software .In Vivo Experiments) guidelines to improve standards of reporting of animal experiments was used. The experiment was divided into four groups: Control group; OA group; Shift group; and Non-shift group.Adolescent (8–10 week old) Sprague Dawley rats were purchased from Liaoning Changsheng biotechnology co.,Ltd. The applicable Johns Hopkins Bloomberg School of Public Health Guidelines for the care and use of animals were followed. The ARRIVE  to the experimental rats was used according to the modeling method summarized by , and took images for analysis. The scoring criteria for OA cartilage pathology were based on the assessment system issued by Osteoarthritis Research Society International (OARSI) .For immunohistochemical analysis, deparaffinzing and rehydrating paraffin sections were performed following by antigen retrieval, blocking endogenous peroxidase activity and serum sealing. Incubations were performed overnight at 4°C with primary antibodies for BMAL1 (1:100), MMP-3 (1:100), MMP-13 (1:100), ADAMTS-4 (1:200), β-catenin (1:100), Aggrecan (1:100), and COL2A1 (1:20) prepared with PBS. Then the tissues were covered with secondary antibody (HRP labeled) and incubated at room temperature for 1.5 h. After Diaminobenzidine (DAB) chromogenic reaction and nucleus counterstaining with hematoxylin stain solution, the slices were dehydrated and sealed following by examined under a microscope  and analyzed.The serum and extracellular secretions (ECS) was extracted after centrifugation . ELISA was performed following the instructions strictly by using rat tumor necrosis factor α (TNF-α) , Interleukin-6 (IL-6) , C-terminal crosslinking telopeptide of type Ⅱ collagen (CTX-Ⅱ) , cartilage oligomeric matrix protein (COMP) , inducible Nitric Oxide synthase (iNOS), prostaglandin E2 (PGE2), and cyclooxygenase-2 (COX-2)  ELISA kit. The absorbance of each tube was measured with a microplate reader at 450 nm reflecting the content and activity. ELISAcalc software was used to calculate the regression equation of the standard curve linear according to the concentration and OD value and four-parameter logistic curve was selected for fitting model.in vivo and mean ± S.E.M in vitro. Unpaired t test was performed between the two groups. When three or more groups are compared, IBM SPSS Statistics 25.0 with one-way ANOVA and Tukey test for post hoc test were used. Graphs were drawn using the GraphPad Prism (version 8). Values with p < 0.05 were considered statistically significant, and p < 0.01 extremely significant.Data are presented as mean ± SD p < 0.01) . Similarly, compared with non-shift rats, the shift group showed a similar upward trend of these inflammatory markers   . By eval< 0.001) . It suggTo assess the effect of CRD on the articular cartilage, histological staining and immunological assays of the tibial plateau were performed. As shown in p < 0.001 and p < 0.0001) and the significant decrease of matrix fibers (Aggrecan and COL2A1) (p < 0.001 and p < 0.0001) in OA cartilage indicated that the inflammatory response of OA cartilage was severe. Interestingly, similar changes in protein expression were observed in the shift group , which suggested that LD cycle disturbance bias the rate of cartilage degradation over matrix synthesis. Both ACLT and CRD significantly reduced the expression of BMAL1 in cartilage (p < 0.05 and p < 0.001), indicating the dysregulation of cartilage clock.Additionally, the significant increase of some hydrolytic proteases  (ft group  (p < 0.0artilage  (p < 0.0p < 0.0001) (p < 0.0001)  , and gen 0.0001) . In vitrml IL-1β . The secml IL-1β . It is sp < 0.0001), and the expression of p-β-catenin was significantly decreased (p < 0.0001), compared with the control group. Similarly, a similar trend occurred after silencing BMAL1 (p < 0.0001) (p < 0.0001), BMAL1-KD also accelerated the phosphorylation of GSK-3β (p < 0.01)  . The pro < 0.01) . Moreove < 0.01) . These iIn vivo, the percentage of β-catenin positive cells in the OA and Shift groups was extremely significantly ascended compared to the control group . This corresponds to the results in vivo. The Wnt/β-catenin pathway specific inhibitor XAV-939 has been shown to be significantly effective in improving an OA-mouse model (p < 0.05 and p < 0.0001) (p < 0.05), but had no obvious effect on CTX-Ⅱ (As can be seen from se model . After c 0.0001) . XAV-939on CTX-Ⅱ . These fp < 0.001 and p < 0.0001). XAV-939 significantly recovered the reduced COL2A1 and Aggrecan mRNA (p < 0.05)  , promoti < 0.05) .in vivo or KEGG enrichment analysis are needed.Exposure to long-term LD cycle phase shifts can lead to immune system disruptions and increased levels of chemicals associated with inflammation and oxidative stress . Disruptin vitro studies targeting the β-catenin signaling pathway.Core clock genes dominate the rhythm balance of tissues, among which BMAL1 can regulate the existence of several other clock genes, and plays a vital role in maintaining the normal function and integrity of body . CartilaApcMin/+ mice (BMAL1−/− mice (in vitro studies also confirmed the results.Wnt/β-catenin signaling pathway, one of the most studied pathways in OA, is closely related to chondrocyte differentiation, proliferation and apoptosis, and plays a crucial role in cartilage catabolism and regeneration . Zhou et/+ mice ; in exud/+ mice . These e/+ mice . Inhibit/+ mice . Various/− mice . Combinein vitro (These present a question that whether the interaction mechanisms of BMAL1 and Wnt pathways are different in different microenvironments? And the Wnt/β-catenin pathway plays a different role? There are several possible explanations for the current contradictory findings. Firstly, The environment of cell proliferation is different. Some injuries, such as mechanical injury OA, compensatory cell proliferation is intense; The Wnt/β-catenin pathway is also significantly activated during vigorous periods of normal or abnormal proliferation of tumor or stem cells. Clock genes may play different roles in different contexts. In addition, the mechanism of Wnt/β-catenin pathway in OA is still unclear. Liu et al. presented a novel therapy in alleviating OA by activating the Wnt/β-catenin signaling pathway . Therefoin vitro .Additionally, we found that long-term CRD significantly accelerated weight gain in rats from Week 10, which was consistent with the results of


By increasing the environmental concerns and depletion of petroleum resources, bio-based resins have gained interest. Recently, lignin, vanillin , and divanillin -based resins have attracted attention due to the low cost, environmental benefits, good thermal stability, excellent mechanical properties, and suitability for high-performance natural fiber composite applications. This review highlights the recent use of lignin, vanillin, and divanillin-based resins with natural fiber composites and their synthesized processes. Finally, discussions are made on the curing kinetics, mechanical properties, flame retardancy, and bio-based resins’ adhesion property. T. Tg rang−1 was merged with the –OH peak of PVA-lignin resin. Moreover, the thermal stability of EPLM resin was slightly higher, around 240 °C at 20 wt% of MA, than the PVA-lignin resin (230 °C) . Mo. Mo−1 wag (132 °C) at a 2:1 epoxy/amine ratio . . g of cuic chain . Moreoveg of the cured TE-APP decreased from 176 to 153 °C with increasing the APP concentration, and it also decreased with increasing the TE content. Moreover, TE0-APP15 resin showed a better FR with LOI value (35.4%) and the UL-940 test. At the total ratios of 10 phr of TE and APP, the LOI values of the TE-APP resin reached up to 29%. On the other hand, the Tg of MP-APP increased from 164 to 176 °C, decreasing MP and APP’s total ratios from 10 to 5 phr. The increased Tg of MP-APP might be due to the cross-link density of polymers [Xu et al.  have synpolymers . Meanwhipolymers ,111. Thepolymers  with TGAs denotes the statistic heat resistant index and Td5% and Td30% are at 5 wt% and 30 wt% loss in TGA. The TGA results demonstrated that the Ts of the TE-APP resin was near the MP-APP resin, while the Ts for TP-APP resin was the lowest. Therefore, TE-APP resin depicted the highest thermal stability among the resins. On the other hand, MP-APP resins showed good thermal stability due to the rich benzene ring. The thermal and FR properties of TP, MP, and TE-based resins are summarized in Here, Td5% and Tmax at 286 °C and 343 °C, respectively. PLA exhibited Td5% and Tmax at 326 °C and 363 °C, respectively. As increasing the wt% of VP, the composite’s thermal stability was found in 324–333 °C. The Tmax increased from 366 to 380 °C. These results suggest that the VP is a considerable additive for plastics. The thermal and FR properties of PLA/VP-based composites are presented in g as 78 °C, while the tan delta exhibited the Tg around 87 °C. The lower Tg of DGEBA-VN2HMDA resin may be due to the flexible segments of the hardener. On the other hand, the resin’s thermal stability also appeared lower (302.9 °C) than the conventional epoxy polymer due to the amine group’s low binding energy. DGEBA-VN2HMDA resin did not dissolve in common solvents in comparing uncured resin, but it swelled and afterward partially dissolved some content in a polar solvent. DGEBA-VN2HMDA has hydrolysis properties due to the imine bond, although it did not change the weight and shape with water immersion.To increase the FR and toughness, Cheng et al.  have prem) of MPPN (130.4 °C) may be attributed due to the rigidity of propargyl content and higher viscosity (0.45 Pa.s) as compared to AMPN, which have Tm = 94.6 °C and viscosity = 0.08 Pa.s. Besides, the gelation temperature of MPPN was 286.2 °C, while that of AMPN was 294.6 °C. DSC revealed the three exothermic peaks after the Tm for both monomers. According to both monomers’ chemical structures, the first exothermic peak told in DSC might be due to the possibility of Claisen rearrangement [−1) disappeared. The band intensities of –CN (at 2228 cm−1) and phthalocyanine (1014 cm−1) were reduced after curing at 375 °C, which means that the curing of AMPN ended the phthalocyanine stage [−1 and 2131 cm−1 related to alkynes stretching were disappeared entirely. The hydroxyl group band did not appear, while the bands of –CN, phthalocyanine, isoindoline (1701 cm−1), and triazine (1350 cm−1) are remarkably reduced. This change indicates that the alkynes were completely rearranged but partially cross-linked. The calculated processing window temperatures for AMPN and MPPN were 185 °C and 148 °C, which is the difference in the gelation temperature and Tm temperature. Moreover, for the VN-based phthalonitrile monomer, this temperature was different from the petroleum-based monomers [E′). However, Tg of the cured phthalonitrile resins was higher than 500 °C, comparable to the petroleum-based polymers. Navarukiene and coworkers [Recently, Han et al.  have synangement . The secne stage ,116. Formonomers . Regardiosity 0.4 Pa.s as oworkers . In the oworkers ,120.g, and Tα of DVN epoxy resin. It found that the yield percentage of tetra-GEDVA to be 90% at 10 eq. NaOH/OH ratios. The thermoset behavior of the cured DVN epoxy resin monitored by DSC was based on the stoichiometric ratio (r), calculated by the equation [g (198 °C) and Tα (200 °C) when r = 1; for di-GEDVA-IPDA, Tg = 138 °C, Tα = 140 °C; for tri-GEDVA-IPDA, Tg = 163 °C, Tα = 177 °C; and for DGEBA-IPDA, Tg = 152 °C, Tα = 155 °C. Tα increased from 140 to 200 °C due to the increased cross-linking with the epoxy content increase. Thus, epoxy networks led to reduced mobility. Nevertheless, the onset temperature of cured GEDVA-IPDA resin was lowest than that of DGEBA-IPDA, which means that GEDVA resin has a high reactivity tendency towards the N-H group. In comparing DGEBA-IPDA, GEDVA-IPDA resin exhibited the higher Young modulus, which increased from 1.45 to 1.9 GPa with the epoxy content increase. On the other hand, elongation at the break of DGEBA-IPDA was higher than those of GEDVA-IPDA resin due to the isopropyl network, which provides more flexibility to the DGEBA segment resin. The thermal degradation behavior of GEDVA-IPDA and DGEBA-IPDA was investigated in the view of Ts by Equation (1). GEDVA-IPDA resin exhibited two types of degradation; the first degradation occurred at 275 °C with 40% weight loss and the second near 500 °C with 60% weight loss, while for DGEBA-IPDA, the first degradation started near 330 °C with 70% weight loss. On the other hand, in N2, GEDVA-IPDA resin revealed the first degradation near 275 °C, while for DGEBA-IPDA near 350 °C. GEDVA-IPDA resin showed a lower Ts value around 20 °C than DGEBA-IPDA, attributed to the –OCH3 group’s degradation in an aromatic ring [DVN is attractive as a sustainable and bio-based polymer derived from renewable resources like lignin. It is also considered a building block for polymer synthesis ,122. Sevequation .(2)r= fnd epoxy . DSC thend epoxy ,134. Curtic ring ,136. DVAg = 204 °C, char residue = 16%), tetra-GEDVA-DMAN exhibited the higher Tg around 212 °C with 48% char yield, which might be attributed to the C-C bonding between two aromatic rings in DMAN.Recently, in this way, Savonnet and coworkers  have rep1c). K1c increased with increasing the fraction of DL-epoxy in the composite. At 1.0 wt% of DL-epoxy fraction, the composite showed the highest K1c near to 1.78 MPa.m1/2. Conversely to neat epoxy, DL-epoxy/cured epoxy exhibited low Tg due to the low cross-linking density at 1.0 wt% of DL-epoxy fraction [g [Commonly, polymeric resins are categorized into epoxy and ester ones, in which ester resins are utilized for low-performance applications while epoxy resins for high performance ,138,139.fraction . The touction [g ,153,154.ction [g . To thisction [g . In thisction [g  have repction [g  have preRecently, Ko et al.  have rep−2 compared to DGEBA (27.3 kJ m−2) because IA fills the gap of the epoxy matrix. Additionally, VNER/EF205/2 wt%-IA demonstrated the jagged and rough surface than those of DGEBA and VNER/EF205. It means VNER/EF205/2 wt%-IA can absorb higher energy, resulting in higher energy to break the materials, which indicates its higher toughness than the DGEBA [g, and char yield of VNER indicated that it could be used in high-performance applications. Xu and coworkers [g of TP-AAP, TE-AAP, and MP-AAP are comparable with DGEBA. Due to the higher char yield, these showed the potential use in high-performance materials. Wang and coworkers [g (172 °C) near the commercial CF/DER331-PACM composite. Therefore, the MB-PACM resin is applicable for the carbon fiber reinforced polymers composites. Nal et al. [2 from 12.42 kJ/m2 that increased may be due to the multifunctional groups of PVSi. Moreover, fracture surface roughness and surface irregular cracks increased with the incorporation of PVSi content, while the neat epoxy demonstrated the flat surface with straight lines cracks. The irregular cracks lead to higher surface energy and plastic deformation energy, resulting in higher toughness [Vanillin is an aromatic bio-based compound, and due to its aromatic character, its epoxy resins have been identified to replace vinyl ester-based resins . Incorpohe DGEBA . This rehe DGEBA  synthesioworkers  have repoworkers  reportedl et al.  synthesil et al.  have recoughness . On the oughness . Besidesoughness ,169. TheLiu and coworkers  preparedg of all composites  found to be comparable with DGEBA. In the FR test, GEDVA thermoset specimens stopped burning rapidly with the char layer while the DGEBA thermoset burned quickly with flame. DVN-based thermosets are promising materials for composites as flame retardant high performance and adhesive applications.DVN is known as a bio-based phenolic compound, and it is used in broad applications such as test enhancers, the food industry, organic light-emitting diode, and nanocomposite materials ,122,172.This review paper elucidated broad research literature and synthetic activities on lignin, VN, and DVN epoxy resins, focusing majorly on curing kinetics and curing agents, thermal stability, adhesion property, and mechanical properties. All recent aspects of lignin, VN, and DVN epoxy materials were reviewed, with perspectives for alternative toughened thermosets for natural fiber composites, FR, and as adhesives for high-performance application.Although commercial epoxy resins are prevalent for various applications, bio-based epoxy resins from lignin, VN, and DVN could be alternates for commercial epoxy resins due to their high thermal stability tensile strength with desirable ecofriendly and environmental safety. Many papers have been published on the lignin, VN, and DVN related to coatings, FR, adhesives, building materials, and high-performance applications. However, limited study has been published on the lignin and VN-based epoxy resins for natural fiber composites, although bio-based DVN epoxy resins are flourishing for natural fiber composite applications. Additionally, DVN can also be devoted to developing bio-based curing agents to reduce environmental concerns. This review explored the utilization of lignin-derived bio-based resin to develop natural fiber/polymer composites with high-performance applications.With more attempts from the worldwide research community, advanced flame retardant and high-performance lignin, VN, and DVN-based epoxy resins might be a technical and commercial success for natural fiber composite applications. This review will increase the research community’s attention to bio-based resins production to reduce the use of petroleum-based resins. The literature depicts limited research progress on resin’s adhesion strength with natural fibers, including natural fiber composites development with lignin-derived resins. However, this literature shows challenges that must be overcome to develop fully bio-based resins, including bio-based hardeners, and enhance resin’s binding adhesion strength with natural fibers to obtain environment-safe high-performance composites. Finally, we are optimistic that combining various tailoring properties and the design methodology will achieve technical and commercial success. Nevertheless, there will be a massive need to improve the resins’ adhesive property and toughness for natural fiber composites.


Motor Imagery (MI) is a powerful tool to stimulate sensorimotor brain areas and is currently used in motor rehabilitation after a stroke. The aim of our study was to evaluate whether an illusion of movement induced by visuo-proprioceptive immersion (VPI) including tendon vibration (TV) and Virtual moving hand (VR) combined with MI tasks could be more efficient than VPI alone or MI alone on cortical excitability assessed using Electroencephalography (EEG).We recorded EEG signals in 20 healthy participants in 3 different conditions: MI tasks involving their non-dominant wrist (MI condition); VPI condition; and VPI with MI tasks (combined condition). Each condition lasted 3 minutes, and was repeated 3 times in randomized order. Our main judgment criterion was the Event-Related De-synchronization (ERD) threshold in sensori-motor areas in each condition in the brain motor area.The combined condition induced a greater change in the ERD percentage than the MI condition alone, but no significant difference was found between the combined and the VPI condition (p = 0.07) and between the VPI and MI condition (p = 0.20).This study demonstrated the interest of using a visuo-proprioceptive immersion with MI rather than MI alone in order to increase excitability in motor areas of the brain. Further studies could test this hypothesis among patients with stroke to provide new perspectives for motor rehabilitation in this population. Vibratory stimulation is already used in various medical applications, such as pain management or proprioceptive rehabilitation after stroke ,2. It haIn the last twenty years, a growing number of studies have taken an interest in developing Virtual Reality tools (VR) in various fields, such as social psychology , haptic Another tool that can be used to stimulate motor areas of the brain is Motor Imagery (MI), which consists in imagining moving the limb without performing any actual movement. MI is already used for upper or lower limb rehabilitation in motor rehabilitation after neurological impairments ,24. It iIn the literature, cortical activations have been evidenced using functional imaging and electroencephalography (EEG) recordings. EEG activity can be recorded during MI or tendon vibration from electrodes placed in the motor areas of the brain. It is then processed to extract relevant features from the recorded signals. Sensory-Motor Rhythms (SMRs) consist in brain wave oscillations generated by the somatosensory and motor cortices and recorded, mainly, from the C3 (left motor area) and C4 (right motor area) electrodes. SMRs are detectable at frequencies of 8–28 Hz ,31. SMR The effects of the combined association of illusion of movement and MI to stimulate cerebral motor areas have not been clearly described to date. The literature suggests some hypotheses, such as the fact that proprioceptive illusions and MI could share the same neural underlying mechanisms . ChatterWe hypothesized that a visuo-proprioceptive stimulation  combined with MI could increase the cortical excitability of brain motor areas compared to MI or visuo-proprioceptive stimulation taken separately, by the production of a virtuous closed-loop feedback. Improving and reaching the best possible cortical excitability could enable optimal brain plasticity and could thus be an efficient means of neuro-rehabilitation for patients.The aim of our study was therefore, using EEG, to evaluate whether a visuo-proprioceptive immersion (VPI) including tendon vibration (TV) and Virtual hand movement  combined with MI tasks could be more efficient than VPI alone or MI alone, in terms of cortical excitability of motor areas of the brain among healthy participants.From October to November 2019, we conducted a single-centre randomized controlled pilot study in the Rehabilitation Unit of Rennes University Hospital in France. The study was promoted by the Rennes University Hospital Center and obtained the approval of the Ethics Committee of Strasbourg University, France, on October 8th, 2019 (registration number: 19/62-SI 19.07.05.46737). An information letter was issued to the participants including: the aims of the study, the protocol, the risks involved and insurance details. Written consent was obtained from each participant prior to testing. This study was recorded in Clinical Trials under the following registration number NCT04130711. No changes to the study design were made after approval by the ethics committee. The individual pictured in  has provVolunteering healthy participants were recruited using a public advertising strategy in the Department of Rehabilitation unit of Rennes University Hospital and of the Medicine Department of Rennes University. A total of 20 healthy participants (Mean± Standard Deviation): 31.30±9.86 years old, Median = 30, Min = 22, Max = 61, Q1 = 24.75, Q3 = 33.25 participated in the study, with 11 males (55%) and 9 females (45%). All healthy participants met the following inclusion criteria: age between 18 and 80 years; no previous history of neurological illness , right-handed. Participants deprived of freedom and with a legal incapacity were excluded from the study. We made this protocol as a pilot study. Concerning the number of participants, the corresponding literature motivating the research hypothesis includes studies involving 15  or 16 paThe participants sat in a typical office chair in front of a computer screen in a quiet room. The EEG cap was positioned on their scalp and the vibratory mechanism on the flexor carpi tendon of their non-dominant arm (left arm), positioned in a shell and hidden from view . This exAfter the MI tasks (in combined or MI condition), a Likert scale from 1 to 7  appeared on screen for the participant to quantify the difficulty in performing the MI tasks. At the end of the experiment, all the participants completed a questionnaire to determine whether they had already tried vibrating devices or MI of this type, to obtain subjective data on vibration comfort, and to obtain information about the illusion of movement felt. We then analysed the ERD values for each condition in order to determine which one was the best to improve cortical excitability and whether visuo-proprioceptive stimulation could trigger motor area activation during MI.After VPI or the combined condition, a Likert scale from 1 to 7 appeared on screen for the participant to quantify the intensity of the illusion .The virtual scene was shown to the participants by using Unity 3.5 software and was composed of a moving, homemade, neutral, white-skinned hand avatar. The scene was displayed on a 17 inch-LCD monitor, the angle of view was that of the virtual avatar and the monitor was positioned in order to match the participant’s first perspective. The movement executed by the virtual hand was an extension of the non-dominant wrist with a total displacement of 30 degrees from resting position, at a speed of 3 degrees per second, congruent with the illusory movement that was expected by the application of a flexor carpi tendon vibration Figs  and 3 [7™ Model 320–105 vibratory unit . EEG recordings were band-pass filtered from 0.5 to 40 Hz and then digitally converted with a sample frequency of 512 Hz (using a Butterworth zero phase filter with a 48 dB slope). All EEG recordings were inspected visually and electrodes with too high impedance >30 kHz) or poor signal quality were excluded from further analysis. Concerning the 20 subjects in the study, 16 of them have intact sessions and 4 of them have one session deleted (one third of a condition). Imagined movement in sensorimotor rhythm paradigms causes ERD in μ (8–13 Hz) and β (13–28 Hz) rhythms, known to be involved in imagining movement, task preparation or active tasks, and observed in the primary motor cortex, contra-lateral to the limb involved in the task, mainly related to C3 and C4 electrodes [ kHz or ptaskE and restE denoting the average power in the frequency range of electrode E during the task condition and at rest, respectively. Positive power changes in strength will be referred to as ERS whereas negative changes will be referred to as ERD. ERD and ERS are percentage values, the reference value being calculated according to the remaining tasks. Results were visualized for the alpha (8–13 Hz) and beta (13–28 Hz) bands as topoplot maps.With The primary outcome measure was the ERD values measured in μ and β bands on the C4 electrodes in each condition . We used C4 electrode as primary outcome because it was estimated that the most important information would come from the right brain motor areas. We then examined other electrodes near C4. The secondary outcome measures were: a) the intensity of the illusion of movement felt during tendon vibration scored on a Likert scale  and b) the difficulty in performing MI tasks (from 1 to 7 with 1 = easy to 7 = difficult). The participants had a computer mouse (with their free right hand) which enabled them to respond on the Likert scale on the screen after each set. The data were collected in the Data Archiving and Networked Services (DANS) databases.2 = 24.47, p<0.001. We therefore used a non-parametric approach (Friedman test and then Wilcoxon signed rank test) to compare mean ERDs on C3 and C4 electrode across condition. We used a two-way repeated ANOVA to compare each conditions  in each band frequency  for all the participants and we tested for an interaction between condition and frequency, then we used a Kruskal-Wallis test to compare the mean ERDs according to subgroups. We also used a Wilcoxon rank sum test to compare data between two conditions. P-values <0.05 were considered statistically significant.Data processing and signal analysis were performed using MATLAB R2017a . The results for the spatial activation patterns are given as the relative power change from baseline ± standard deviation (SD). Topographical plots were created using a customised MATLAB function. We used percentages to describe qualitative variables, and mean ± standard deviation to describe quantitative variables for parametric data. We also used median and interquartile intervals to describe non-parametric data. All data were analyzed by statistical tests using R and MATLAB software. According to the Shapiro-Wilk test, our data followed a normal distribution . According to Mauchly’s Test of Sphericity, our data set violated the assumption of sphericity, for the main judgment criterion: χThis is the flowchart of the experiment .2 = 24.47, p<0.001) according to the ERD percentage. There was no significant difference between the combined condition and the VPI condition (p = 0.59), but there was a significant difference between the combined condition and the MI condition (p<0.001) and between the VPI condition and the MI condition (p<0.001).The mean ERD (C4 electrode) values in percentages ± Standard Deviation were 38.00±10.08 for the combined condition, 33.04±9.93 for the VPI condition and 13.34±12.08 for the MI condition . Using a2 = 17.20, p<0.001) according to the ERD percentage. There was a significant difference between the combined condition and the VPI condition (p<0.01), between the combined condition and the MI condition (p<0.001) but there was no significant difference between the VPI condition and the MI condition (p = 0.06).The mean ERD (on C3 electrode) values were 19.88±12.16 for the combined condition, 7.06±23.88 for the VPI condition and -3.67±28.89 for the MI condition. Using a Friedman test, there were significant differences across the 3 conditions  and we also found a significant result depending on the frequency band . Nevertheless, we did not find significant differences by comparing the VPI and MI condition on C4 electrode using this method  for the conditions analysis nor for the frequencies analysis. We have also tested for an interaction between frequency and condition. We performed the test by comparing each condition to the others . There was no significant difference in the results: between the MI condition and VPI condition , between the MI condition and combined condition , between the VPI condition and combined condition . Then, by using a Kruskal-Wallis test, we found that the combined condition was significantly superior to the MI condition in the μ band, ß band and μ + ß band for all the brain motor electrodes . Comparing the VPI and MI condition, we found only a significant difference concerning the FC2 electrode. Similarly to our previous analyses, we did not find any significant difference between the combined condition and the VPI condition.According to the data, the topoplots represented the power of ERD measured in the 8–28 Hz bands (μ and β bands) around the scalp averaged across all the participants in the study .Concerning C4 electrode, among the participants who preferred to perform the MI tasks during vibration (n = 9), the ERD average value was 41.85% ± 11.11, while in the population of participants who found MI more difficult to perform when visuo-proprioceptive stimulation was present at the same time (n = 11), the result was 34.86% ± 8.41. There was no significant difference between the two groups . The groups were defined following the subjective reports. The participants evaluated the difficulty to perform MI during tendon vibration by using a Likert scale from 1 (very difficult) to 5 (easy to perform).Among the 5 participants who had previously performed MI tasks (see subjective reports), the ERD value was 5.82% ± 27.45 in the MI condition, differing from the other participants who had never performed it before 16.05% ± 6.94. There was no significant difference between the groups .We did not find any correlation between the intensity of the illusion of movement felt by the participants and the level of cortical activation represented by the ERD in the VPI condition  or in the combined condition .2 = 12.77, p<0.01) according to the PSD values. There was no significant difference between the combined condition and the VPI condition (p = 0.24), but there was a significant difference between the combined condition and the MI condition (p<0.01) and between the VPI condition and the MI condition (p<0.001). We compared the values in each condition between the task and the corresponding resting period: we found a significant difference in each condition: Combined condition, VPI condition and MI condition (p<0.001).A power spectrum density (PSD) analysis showed the evolution of the signal power according to the frequency bands of interest in the 8–28 Hz range on the C4 electrode, according to each condition and in comparison with the resting state . There wA time-frequency analysis was performed for each condition, with the average results of all study participants averaged over the 10-second period of stimulation in the 8 to 28 Hz frequency bands .The median  for Likert ratings was respectively 6  for the VPI condition and 5  for the combined condition. There was no significant difference across conditions .The intensity of illusion of movement was also measured across time according to the Likert ratings. For each condition, we analyzed the Likert scores measured at the start of the experiment compared to the values at the end of the experiment. The median  was 5  (in session 1 of the condition i.e. the first 3 recorded minutes) and then 6  (in session 3 of the condition i.e. the last 3 recorded minutes) for the VPI condition. There was no significant difference between sessions 1 and 3 . The median  was 5  (in session 1 i.e. the first 3 recorded minutes) and then 5  (in session 3 i.e. the last 3 recorded minutes) for the combined condition . There wThe median  Likert ratings were respectively 4  for the MI condition and 4  for the combined condition. There was no significant difference across conditions .The perceived aptitude to perform the MI tasks was also measured according to time following the Likert rating. For each condition, we analyzed the Likert ratings measured at the start of the experiment compared to the values at the end of the experiment. The median  was 4.5 (in session 1 of the condition i.e. the first 3 recorded minutes) and then 4  (in session 3 of the condition i.e. the last 3 recorded minutes) for the MI condition. There was no significant difference between sessions 1 and 3 . The median  was 5  (in session 1 i.e. the first 3 recorded minutes) and then 4  (in session 3 i.e. the last 3 recorded minutes) for the combined condition . There wAmong our 20 participants, 5 participants (25%) had already performed MI tasks at least once. Most of our participants felt tired at the end of the experiment , but 16 participants (80%) thought that there was sufficient resting time during the protocol. Nine participants (45%) thought that their MI performance was better and easier when the tendon vibration was applied simultaneously.The main aim of our experiment was to evaluate whether a visuo-proprioceptive stimulation including an illusion of movement induced by tendon vibration, a VR environment and MI tasks could be more efficient in terms of cortical excitability in sensorimotor areas than either MI tasks or visuo-proprioceptive stimulation alone.First, the results demonstrated that the combined condition was significantly better than the MI condition in terms of ERD values for motor area of the brain. However, we did not find any significant difference between the combined condition and the VPI condition, and between the VPI condition and the MI condition, considering the two-way repeated ANOVA. In time-frequency analyses, we observed greater ERD peaks in the combined condition than in the other conditions, in the μ bands 8–13 Hz), within the stimulation time . In powe–13 Hz, wOne hypothesis that could explain the lack of evidence of superiority of the combined condition compared to the VPI condition is the possible absence of physical accumulation of ERD from different origins, and the difference in the behaviour modulation of ERD/ERS. Rimbert et al. showed that combined MI and electrical stimulation modulated the generation of ERD and ERS differently from MI alone or electrical stimulation alone. The ERDs did not seem to accumulate whereas the ERS were significantly amplified in the same condition . The worIn the vibration condition , we founIn our study, 9 participants (45%) thought that their MI performance was better and easier when the VPI was applied simultaneously with their MI tasks, while the others felt that it was disturbing. This subjective result could be explained by the positive effect of repeated tendon vibrations inducing illusory movement while the participant were performing MI tasks to enhance their MI performance, as noted by Yao and al. (2015) . In thisWe found that the sensation of illusion induced by tendon vibration was constant throughout the time of the experiment, without any habituation among the participants . By usinHowever, our study presents some methodological limitations. First, we included a small population of 20 healthy participants. We only found in the existing literature 2 studies with a similar topic and methodology and none of them seemed to perform a power calculation ,37. We cIt could be interesting in further studies to test the same hypothesis for patients with stroke to see if similar results are obtained. This population is often older  than theBCI studies can use MI as a substrate, and there is a wide corpus of literature on the haptic feedback used . VibratoIn conclusion, we found that visuo-proprioceptive stimulation and MI could be efficient to stimulate brain motor areas. VPI is an attractive and interesting tool to use in BCI systems, with MI. Overall, our results pave the way for the design of new tools in rehabilitation using VR and haptic stimulation associated with a brain-computer interface.S1 Checklist(DOCX)Click here for additional data file.