Regardless of set , conditions 1 and 2 produced higher values of anaerobic capacity (p ≤ 0.05). Similarly , condition 1 showed higher wheel cadence (p < 0.02) than condition 3. A bilateral joint cooling for 20 min with or without pre-cooling warm-up may improve overall sprint cycling capacity during five sets of cycling intervals when compared to the non-cooling condition.We compared the effect of bilateral knee joint cooling with or without a pre-cooling warm-up on sprint cycling performance to a non-cooling control condition. Seventeen healthy young males  performed three conditions in a counterbalanced order . For warming, a single set of cycling intervals  was performed. For cycling, five sets of cycling intervals were performed. For cooling, 20 min of bilateral focal knee joint cooling was applied. Peak and average values of anaerobic capacity and wheel cadence during each set across conditions were statistically compared. There was no condition effect over set (condition × set) in anaerobic capacity (F Cold application—extracting heat and energy (reducing temperature)—is an inexpensive and convenient way to achieve clinical benefits such as metabolic reduction  and anesBased on the observation of improved sprint ability , we coulTherefore, the purpose of this study was to examine the effect of 20 min of bilateral knee joint cooling with or without a pre-cooling warm-up activity on sprint cycling performance. Specifically, we asked: (1) will knee joint cooling result in an improvement in anaerobic capacity and wheel cadence, compared with a non-cooling condition? and (2) if so, will the joint cooling conditions with pre-cooling warm-up show better cycling performance than the joint cooling condition without pre-cooling warm-up? We hypothesized that (1) conditions with knee joint cooling would show better cycling performance than a non-cooling condition, and (2) there would be no difference in cycling performance between the conditions with or without pre-cooling warm-up. The results of this study would directly assist cyclists and coaches by suggesting necessary components for warm-up activities prior to racing. Additional measurements in thigh muscle temperature, fatigue perception, and heart rate during five sets of cycling protocols would also help us explain the cause-and-effect relationship between warm-up activity with knee joint cooling and cycling performance.Our sample size was calculated based on the primary dependent measurement, the anaerobic capacity of cycling sprint (watt/kg). In a similar ergometer test, the average values on healthy adults were between 8 and 10 watt/kg . To deteTherefore, seventeen healthy young males  participated in this study. Subjects were physically active (participating in at least 150 min of aerobic exercise per week) and free from cold urticaria, lower-extremity or back injury in the last six months, and lower-extremity or spine surgery in their lifetime. Subjects with any known disease or disorder  were excluded. Prior to participation, each subject read the testing procedures and provided informed consent, approved by the University’s Institutional Review Board (KHGIRB-19-141).To experience three conditions, subjects reported to the laboratory at the same time for three visits, at least two days apart. Subjects were asked to keep their habitual diet and not to participate in any physical activity for 24 h prior to the visits . The rooOn the first day of data collection, subjects gave informed consent. Upon arrival to the laboratory for each visit, subjects had a 5 min supine rest on a treatment table. During this resting period, the baseline values of the thigh temperatures, fatigue perception, and heart rate were recorded. Afterward, one of three conditions , thigh temperatures , fatigue perception , and heart rate (using a heart rate monitor).http://www.goldencheetah.org/ accessed in 11 November 2019). Average and peak values during each set of 10 s cycling sprints (except for the first set for condition 1) were compared.To test cycling performance, peak and average capacity and wheel cadence during a 10 s cycling sprint during the last five sets of cycling protocol were compared across conditions . Using the ergometer’s built-in computer, data were sampled at 50 Hz for anaerobic capacity (watt) and wheel cadence (revolution per min: rpm) and then wirelessly exported into a spreadsheet program using software  using two separate channels of thermistor probes were used D. Each tThe heart rate monitor  was wirelessly connected to a mobile phone application (Polar beat: Run & Fitness). Heart rate (bpm) data were then exported into the spreadsheet. The values at the same time points as the thigh temperature samples were compared.Room temperature and relative humidity were recorded using a wireless environmental data logger .p ≤ 0.05 for all tests; SAS 9.4: SAS Institute Inc., Cary, NC, USA). Cohen’s d effect sizes (d) [To test the condition effect over set, we performed a 3 (condition) × 5 (set) mixed model ANOVA for anaerobic capacity and wheel cadence; a 3 (condition) × 6 (baseline and each set) mixed model ANOVA  for thigh temperature, fatigue perception, and heart rate. Tukey–Kramer pairwise comparisons were used for post hoc tests (izes (d)  were cal8,224 = 0.72, p = 0.67) or peak  and peak  values in anaerobic capacity were reduced at set 4  and 5 , respectively, as compared with those at set 1.There was no condition effect over set (condition × set) in average  and peak cadence , condition 3 showed 5  and 4  fewer wheel rpm as compared with conditions 1 and 2, respectively , subjects with condition 1 had 3 greater wheel rpm than those with condition 3 , the average cadence between sets 2 and 5 was different . The highest peak cadence  was recorded at set 2, as compared with sets 1 , 4 , and 5 .There was no condition effect over set (condition × set) in average cadence . The VM temperature under condition 2 was 0.7 °C lower than condition 1′s temperature . The VL temperature for condition 2 (32.5 °C) was lower than the temperature at conditions 1  and 3  and 3 .There was also a condition effect over set in the VL temperatures , fatigue perception for condition 3 (4.7 cm) was 19% higher than conditions 1 or 2 , subjects began to feel fatigue at set 3  as compared to the baseline (2.8 cm).There was no condition effect over set in perception of fatigue , heart rate for condition 3 (138 bpm) was 2% higher than for conditions 1 or 2 , subjects’ heart rate immediately increased at set 1  and then further increased at sets 3  and 5  from the baseline value (76 bpm).There was no condition effect over set (condition × set) in heart rate , our hypotheses that the conditions with joint cooling, regardless of the pre-cooling warm-up, would show a better cycling sprint performance than a non-cooling condition and that there would be no difference between the cooling conditions were generally accepted. Therefore, the order of cooling and warming up does not seem to be an important factor. We did not observe the peak cycling performance on a specific condition at a specific set during cycling interval protocols. When collapsing data across time points (condition effect), knee joint cooling appears to be beneficial in cycling performance in terms of anaerobic capacity and cadence. Our results, therefore, indirectly support the previous data on physical performance enhancement involved with pre-exercise cooling ,5,16,17.Statistically, the performance enhancement in cycling sprints was not observed in an interaction (condition × set) but in a condition main effect. Subjects with knee joint cooling (conditions 1 and 2) showed 4 to 5 greater wheel revolutions while completing the five sets of 10 s cycling sprints when compared with those without cooling (condition 3). While the recorded total wheel cadence ranged between 130 and 145 rpm  in our study, the results of the current study indicate that subjects who receive knee joint cooling may generate 4 to 5 additional rpm. The wheel circumference of the stationary bike is 2 m (by manufacture), and our results can be further interpreted as being located 8 to 10 m ahead. Although the standardized mean differences in anaerobic capacity and wheel cadence are small (d < 0.39), relative to the previous data on performance enhancement [While a previous study reported a skin temperature reduction of 23 °C at the knee joint due to 20 min of focal knee joint cooling , assessmMuscle temperature significantly impacts athletic performance ,27,28,29There are several limitations and assumptions in our study. First, the generalization of knee joint cooling on cycling performance should be restricted to the specific situation where subjects performed five sets of cycling sprint intervals; thus, care should be taken when applying the results of our data to different types of cycling performance such as long-distance road cycling. Additionally, our results on a stationary ergometer cannot be directly applicable to road cycling because our testing protocol did not account for additional weights , road condition , and environmental condition . Neurological adaptation to temperature change  may vary between subjects due to physiological and psychological responses to a combined effect of cycling sprints and cold and/or personal experience of cold application and stationary cycling. Even within subjects, the results of our interventions could have been influenced by certain contributing factors such as lab conditions , nutritional and hydration status, and maximal effort. Although heart rate and thigh temperature were collected simultaneously (the same sampling frequency), the recorded values of two variables at the same time points might have not been correspondent due to the heart rate lag . FinallyA 20 min bilateral joint cooling with or without pre-cooling warm-up prior to cycling protocols seems to improve overall cycling performance during five sets of cycling interval protocols, as compared to the non-cooling condition. When compared to the non-cooling condition, the conditions with knee joint cooling showed four to five higher wheel revolutions with 19% and 2% less fatigue perception and heart rate, respectively. The direction of the results in each dependent variable clearly demonstrates an advantage of focal knee joint cooling on cycling sprint performance. We recommend coaches and athletes consider tying knee joint cooling on their training regimens.


E-cadherin in mouse SPCs showed no obvious effect on SPCs homing and spermatogenesis.Cadherins play a pivotal role in facilitating intercellular interactions between spermatogonial progenitor cells (SPCs) and their surrounding microenvironment. Specifically, E-cadherin serves as a cellular marker of SPCs in many species. Depletion of E-cadherin in germ cells was shown to promote SPCs differentiation, evidencing by reduced PLZF+ population and increased c-Kit+ population in mouse testes. E-cadherin loss down-regulated the expression level of β-catenin, leading to the reduced β-catenin in nuclear localization for transcriptional activity. Remarkably, increasing expression level of Cadherin-22 (CDH22) appeared specifically after E-cadherin deletion, indicating CDH22 played a synergistic effect with E-cadherin in SPCs. By searching for the binding partners of β-catenin, Lymphoid enhancer-binding factor 1 (LEF1), T-cell factor (TCF3), histone deacetylase 4 (HDAC4) and signal transducer and activator 3 (STAT3) were identified as suppressors of SPCs differentiation by regulating acetylation of differentiation genes with PLZF.Here, we investigated the regulatory role of E-cadherin in regulating SPCs fate. Specific deletion of Two surface markers of SPCs, E-cadherin and Cadherin-22, synergically maintain the undifferentiation of SPCs via the pivotal intermediate molecule β-catenin. LEF1, TCF3, STAT3 and HDAC4 were identified as co-regulatory factors of β-catenin in regulation of SPC fate. These observations revealed a novel regulatory pattern of cadherins on SPCs fate.The online version contains supplementary material available at 10.1186/s13578-022-00880-w. In their differentiation hierarchies, As (SPCs) . A transs (SPCs) , 3, and s (SPCs) , through) [c-Kit , Sall4 [t [Sall4  and Redd4 [Redd1 . Based ond Dmrt1 .SPCs fate is regulated by their microenvironment through interacting with neighboring cells. And many membrane or transmembrane proteins, such as cadherins, integrins, claudins, are intimately associated with intra- and intercellular functions such as adhesion, binding, recognition and signal transduction , 9. AmonHdac2, Hdac6, and Sirt1 increased, while Hdac8, Hdac9 and Sirt4 decreased, during SSCs differentiation or aging [β-catenin functions as a pivotal intermediate molecule in Wnt signaling pathway , 23. Howor aging . Moreoveor aging . Thus, iHere, the role of E-cadherin in SPCs was studied using conditional knockout mice and in vitro cell culture models. The dynamic role of β-catenin in regulating SPCs fate was identified, and the interaction regarding downstream genes or cooperators was further analyzed. Briefly, E-cadherin was identified as an essential transmembrane molecule to maintain undifferentiated state of SPCs, and CDH22 played a similar role as E-cadherin and might compensate for E-cadherin loss. Deletion of E-cadherin disturbed the dynamic balance of β-catenin in structural maintenance, protein degradation and nuclear localization, leading to a reduced β-catenin expression and promoted SPCs differentiation. Moreover, the interaction among β-catenin, PLZF and HDAC4 was discussed in SPCs. These observations indicated a new regulatory pattern of SPCs differentiation.+ cells residing in the first layer in seminiferous tubules and found to regulate SPCs fate  were generated by mating E-cadherin floxed females with E-cadherinL/+;Ddx4-Cre+ males  self-renewal [E-cadherin loss in SPCs. As shown in Fig. Cdh22 expression in SPCs and confirmed the reduced phosphorylation levels of S33/S37/T41 and S675 of β-catenin . Conversely, knockdown of Tcf3 caused up-regulation of PLZF  to maintain SPCs undifferentiated state, which requires further investigation.Due to lack of DNA binding domain, β-catenin needs to bind to TCF family including LEF1, TCF1, TCF3 and TCF4 in mouse and human to regulate target gene expression . Using Rymocytes , a similHdac1-9 mRNA , but the expression of PLZF was suppressed, and the expression of differentiation markers including c-Kit, STRA8 and SOHLH2, were up-regulated  and regulating acetylation  in gene deletion, the un-infected SPCs populations seemed to be able to reconstitute germ cell pool afterwards. In our study, LoxP-Cre system was employed to enable a conditional knockout, in which the germline specific deletion of E-cadherin started at embryonic stage driven by Ddx4-Cre. Compared to the in vitro deletion system mediated by adenovirus, Ddx4-Cre exerts deletion as early as embryonic day 15 with a more than 95% efficacy [+ population was reduced in E-cadherin deletion group. Overall, these observations implied that E-cadherin might affect SPCs maintenance under physiological conditions. Also, the compensation effect of CDH22-β-catenin complex on SPC development predicted by Shinohara was verified in our study, indicating a delicate interaction between cadherins and Wnt signaling pathway.The complicated interaction pattern between E-cadherin and Wnt signaling pathway shadows our understanding in regulation of SPCs fate. E-cadherin is negatively regulated by Wnt signaling , while Wnization . Using aefficacy . NotablyE-cadherin in germ cells promoted differentiation, but deletion of E-cadherin in SPCs through CRISPR/Cas9 not only gave rise to differentiation, but also reduced anti-apoptosis capacity. We proposed that the testicular microenvironment protected SPCs from apoptosis after E-cadherin deletion, and SPCs in vitro lacking of the protective function of niche were susceptible to apoptosis. However, further experimental evidence is required to verify this hypothesis.To understand the biological role of E-cadherin in regulating SPCs fate, it’s essential to focus on the two major functions of E-cadherin, the structural role and signaling role. In this study, we demonstrated that conditional disfunction of E-cadherin led to a remarkable decrease of β-catenin expression and a consequent decline of translocation to nucleus for transcription. As the pivotal molecule of cadherins and Wnt signal pathways, β-catenin is involved in the regulation of SPCs fate through transcriptional activity. HDAC4 and STAT3 were identified as two novel partners of β-catenin in this study, but the role of Wnt secreted by the niche is not determined, yet. Evidence demonstrated that Wnt5a secreted by feeder cells supports SSC self-renewal through β-catenin-independent pathway [More importantly, we revealed that loss of  pathway , and Wnt pathway . Thus, i+ undifferentiated spermatogonia [+ SPCs population to differentiate into NGN3+ population [β-catenin caused reduced number of PLZF+ and c-Kit+ cells in seminiferous tubules without significant difference in the number of GFRA1+ cells [al and differentiating spermatogonia, due to the fact that GFRA1+ cells primarily distributes in As and Apr [al and differentiating spermatogonia (c-Kit+) populations, rather than inducing SSCs differentiation. Meanwhile the other group claimed that β-catenin promoted differentiation of SPCs instead of affecting self-renewal, supported by the observation that deletion of β-catenin did not change the number of GFRA1+ cells, but rather affected differentiation from GFRA1+ to NGN3+ population, and activation of β-catenin led to GFRA1+ cells loss [al and differentiating spermatogonia. However, the ID4+ cells, a real SSCs enriched population [E-cadherin down-regulated expression of PLZF and GFRA1 in SPCs, indicating that E-cadherin affected the fate of SPCs, including As, Apr and Aal spermatogonia. We noticed that the PLZF+ population highly overlapped with β-catenin+ cells, and disturbance of either PLZF or β-catenin led to up-regulation of differentiation markers represented by c-Kit. Although no direct binding was detected between PLZF and β-catenin, they were found to bind to HDAC4 separately. Molecular assays further demonstrated that HDAC4 combined with β-catenin to suppress differentiation and promote proliferation, and synergically cooperated with PLZF to regulate acetylation of c-kit and Stra8 genes in SPCs.Moreover, the connection of β-catenin and SSCs fate is not fully revealed. Studies from different groups reported controversial functions of β-catenin: to promote the proliferation of PLZFatogonia , or to spulation . However1+ cells , indicat and Apr . Therefolls loss . Collectpulation , were noE-cadherin in germline through LoxP-Cre system caused the down-regulation of β-catenin and up-regulation of AXIN2 [Figs. Axin2 should be positively correlated to transcriptional activity of β-catenin. However, it is worth noting that that E-cadherin could simultaneously activate multiple signaling pathways [Axin2. Moreover, although β-catenin is the intermediate molecule of Wnt and E-cadherin signaling pathways, the target genes activated by Wnt ligands or by E-cadherin loss might be different. Thus, the underlying mechanism is worthy to be explored in future study. Another question is that the connection of β-catenin and other members of cadherin family in germline is largely unknown, yet. Undifferentiated SPCs and round spermatids are likely to be distinct Wnt signaling responders [Cdh22 caused a slightly increase in E-cadherin expression  were purchased from Jackson Lab. The protocols for breeding, mating, and genotyping of E-cadherin floxed mice were identical to previous study [The CD-1 mice for experiments were supplied by Comparative Medicine Centre of Yangzhou University. The us study . All the+ fraction was collected and cultured on mitotically inactivated mouse embryonic fibroblast (MEF) feeder layer at 37 ℃ with 5% CO2. Preparation of culture medium for SPCs and making of MEF feeder cells also followed with previous protocols. SPCs could be stably maintained on MEF feeder layers for more than 30 passages.Testicular cells were extracted from 5 days postpartum mice generally following previous protocol . Briefly−△△CT method, and housekeeping gene Gapdh was used to control the variability in expression levels. The information of primers was listed in Additional file For RNA extraction, tissue or cell samples were treated with TRNZol , and cDNA was reversed-transcripted using GoScript™ Reverse Transcription System . RT-qPCR was performed using TB Green premix Ex Taq II  according to the instruction. Reactions were run in triplicate in three independent experiments. The results were analyzed using the 22O2 treatment. After blocking with 5% goat serum, the slides were incubated with diluted primary and biotin labeled secondary antibodies, respectively. Streptavidin-HRP  and DAB kit  were used for visualization.The protocol for IHC is identical to previous study . BrieflyFor cell IF staining, primary SPCs (within 5 passages) were carried out as described . For memHdac4 or Plzf were amplified and cloned into the pcDNA 3.1(+) plasmid, named as pcDNA 3.1(+)/Hdac4 or pcDNA3.1(+)/Plzf. For luciferase plasmid, c-Kit or Stra8 promoter gene sequences were amplified from genomic DNA using PCR, and inserted into the pGL3 basic plasmid with T4 DNA ligase . There plasmids were nominated as pGL3 basic/c-Kit or pGL3 basic/Sta8 promoter. Both of amplified sequences in the recombinant plasmids were confirmed by sequencing. Primer sequences were listed in Additional file For HDAC4 and PLZF overexpression plasmid, the open reading frame (ORF) of  Takara, 011A. TheFor transfection and CRISPR/Cas9 assays, SPCs of 10–15 passages were used. SPCs were transfected with siRNA or plasmids using lipofectamine 3000  according to the manufacturer’s instructions. The sequences of siRNA used in this study were listed in Additional file Cdh1 were generated using CRISPR/Cas9 approach. SgRNAs designed using the CRISPR/Cas9 Tool (http://crispr.mit.edu) were cloned into pSpCas9(BB)-2A-Puro (PX459). The lentivirus packaging system for infection was . 293T cells were transfected with these four plasmids using Lipofectamine 3000 , and lentivirus in the supernatant was harvested 72 h later and concentrated with a GML-PCTM kit . We performed virus titer assay and infected SPCs following previous protocol [SPCs with targeted knockout in protocol . WesternA-Puro PX59. The lThe protocols for WB and Co-IP are identical to previous study , and detProtein samples separated by SDS-PAGE were electro-transferred to PVDF membrane, which were subsequently blocked in 5% skim milk at room temperature for 1 h and then incubated with primary antibodies overnight at 4 ℃. After that, the membranes were rinsed in TBST followed by incubated with goat anti-mouse IgG-HRP  or mouse anti-rabbit IgG-HRP . Finally, samples were visualized using enhanced chemiluminescence . The information of antibodies was listed in Additional file All procedures were conducted at 4 °C to preserve the protein integrity. Cells were lysed in lysis buffer  with gentle rocking. After centrifugation, the supernatant was collected and transferred to new tubes. Thereafter, the supernatant was incubated with the diluted antibodies overnight with a gentle rotation. Protein A/G agarose beads  were added into the supernatant and incubated for 3 h. Afterwards, beads were precipitated by centrifugation and washed five times with the cold lysis buffer. Finally, the pellet was resuspended in 1 × SDS loading buffer followed by western blot analysis.E-cadherinL/L mice and three 90-day E-cadherinL/L;Ddx4-Cre+ mice were sacrificed to collect testes, and each testis was separately treated for Western blot analysis. For cell samples, SPCs of 3–5 wells in 24-well plates were harvested (around 10^6) for analysis, and all experiments repeated at least three times.For analysis of samples from mouse testes, three 90-day Kit promoter, 0.25 μg of empty pcDNA 3.1(+), pcDNA 3.1(+)/Hdac4 or pcDNA 3.1(+)/Plzf and 3 ng of an internal control Renilla luciferase assay vector pRL-CMV were transfected into HEK 293T cells. Cells were pre-seeded in 24-well plates at a concentration of 4 × 104 per well. Twenty-four hours post-transfection, luciferase activity was measured with a dual luciferase kit  by the Glomax® 20/20 luminometer . Three wells were prepared for each experiment, and the experiments were repeated for three times. The results were calculated by normalizing the luciferase fluorescence value to that of renilla fluorescence.The dual luciferase reporter assay was performed in triplicate based on a previous protocol . Brieflyt-test, *p < 0.05; **p < 0.01.For cell counting, sections or immunofluorescent visual fields were selected randomly. All the spermatogonia in 200× magnification view of microscope were counted and statistically analyzed, and the ratio of SPCs number in knockdown group/control group was defined as relative SPCs number. For IHC, the positive cells in twelve seminiferous tubules of slides were counted under microscope. The seminiferous tubules were randomly selected in discontinuous slides from three mice. Values plotted were expressed as mean ± standard deviation (SD). Statistical analysis was performed using Graphpad prism7 and Student’s Additional file 1: Figure S1. Determination of E-cadherin, β-catenin, Axin2 and ZO-2 in SPCs. The expression of PLZF (A), E-cadherin (B), β-catenin (C), AXIN2 (D) and ZO-2 (E) was detected in purified SPCs using IF staining. Scale bar = 20 μm.Additional file 2: Table S1. Information of primers used in this study.Additional file 3: Table S2. Antibody information in this study.Additional file 4: Table S3. The sequences of siRNA and sgRNA used in this study.


Virtual reality (VR) exergaming is a new intervention strategy to help humans engage in physical activity to enhance mood. VR exergaming may improve both mood and executive function by acting on the prefrontal cortex, expanding the potential benefits. However, the impact of VR exergaming on executive function has not been fully investigated, and associated intervention strategies have not yet been established.This study aims to investigate the effects of 10 minutes of VR exergaming on mood and executive function.A total of 12 participants played the exergame “FitXR” under 3 conditions: (1) a VR exergame condition  in which they played using a head-mounted display, (2) playing the exergame in front of a flat display (2D-EX), and (3) a resting condition in which they sat in a chair. The color-word Stroop task (CWST), which assesses executive function; the short form of the Profile of Mood States second edition (POMS2); and the short form of the Two-Dimensional Mood Scale (TDMS), which assess mood, were administered before and after the exercise or rest conditions.11=3.69, P=.003) as well as the TDMS arousal  and vitality scores , suggesting that VR exergaming enhanced mood. Conversely, there was no effect on CWST performance in either the 2D-EX or VR-EX conditions. Interestingly, the VR-EX condition showed a significant positive correlation between changes in CWST arousal and reaction time . This suggests that the effect of exergaming on improving executive function may disappear under an excessively increased arousal level in VR exergaming.The VR-EX condition increased the POMS2 vigor activity score (rest and VR-EX: tOur findings showed that 10 minutes of VR exergaming enhanced mood but did not affect executive function. This suggests that some VR content may increase cognitive demands, leading to psychological fatigue and cognitive decline as an individual approaches the limits of available attentional capacity. Future research must examine the combination of exercise and VR that enhances both brain function and mood. Many previous studies support the benefits of physical activity for improving physical and mental health. Physical activity has been shown to decrease the risk of noncommunicable diseases, such as cardiovascular disease and type 2 diabetes , and to Recently, exergaming using virtual reality (VR) has been the focus of a new approach to promote physical activity . The maiTo examine the effects of acute exercise under VR on mood and executive function, an experimental model with high reproducibility is required to assess mood and executive function. In this study, we used the color-word Stroop task (CWST) to assess executive function, and the Two-Dimensional Mood Scale (TDMS) to assess mood state . Previous studies have used these measures to examine exercise effects, and have found that 10 minutes of low- or moderate-intensity exercise enhanced mood states, such as arousal and pleasure, and executive function ,25. FurtPrevious studies suggested that 10 minutes of exercise enhances executive function ,25,28-30Prior to beginning the main experimental process, each participant was given verbal instructions, guided through the consent process, practiced the CWST 3 times, and engaged in VR exergaming. Participants were instructed to play the in-game tutorial to learn how the game is played. Once the tutorial was completed, participants played the 10-minute program used in this experiment once to familiarize themselves with exergaming.A few days after the first visit, participants engaged in 1 of the 3 experimental conditions—rest, exercise in front of a display condition (2D-EX), or exercise with an HMD (VR-EX). All participants completed all 3 conditions, each on a separate day, with the order counterbalanced across participants D. In thef=0.50 and paired t test differences exceeding d=0.85 (with a 2-tailed α), as computed using G*Power .A total of 13 right-handed Japanese-speaking young adults (7 men and 6 women) were recruited from June to July 2021. The sample size was determined by assuming that the effects of exercise would be similar to those found in our previous studies ,31. All The FitXR exergame (developed by FITAR LIMITED) was administered using a commercially available HMD . FitXR was selected because it is easy to play even for users with no previous exposure to VR, and can be continued for 10 minutes without interruption even if a play error occurs during the game. Using motion tracking, FitXR simulates handheld controllers as boxing gloves, whereby users punch the targets and must actively crouch and dodge obstacles. The game immerses the participant through tactile sensations of punching the target, auditory sensations of music playing, and performance feedback. In the VR-EX condition, participants played FitXR using an HMD and handheld controllers A.For the same exercise condition without VR exposure , participants exercised while watching the exergaming program movie displayed on a flat screen  positioned 1.5 m away from them B. This aThe CWST, created from web-building platforms (Lab.js v19.1.0) , was useThe CWST comprised 3 trials: 16 neutral, 16 congruent, and 16 incongruent. Each task was presented using the same method as in our previous study ,35, and Heart rate (HR) was measured with an HR sensor  and an HR monitor . Spontaneous eye blink rate (sEBR) was measured as a noninvasive brain dopaminergic system indicator -39. PartParticipant Rating of Perceived Exertion (RPE)  was recoThe POMS2 contains 35 items and evaluates 7 mood states . This study used a subset of 10 items related to vigor-activity and fatigue-inertia to verify whether exergaming enhances mood or causes fatigue.The TDMS  is a momt test (paired) with Holm correction. To clarify the relationships between parameters and executive performance, we conducted Pearson correlation analyses. Statistical significance was set a priori at P<.05 for all comparisons.All analyses were performed using R software  and the EZR on R Commander package . Two-wayThis study was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of the Niigata University of Health and Welfare, Niigata, Japan . Participants were informed that their data would be kept confidential and provided written informed consent to participate in the study.All participants performed the experiment perfectly; no participant reported VR-related adverse effects such as motion sickness, dizziness, and headaches after VR-EX.F2,22=41.9, P<.001; RPE: F2,22=39.5, P<.001) and a significant main effect of time  in sEBR. The paired t test with Holm correction showed significant differences in HR between the rest and 2D-EX conditions, and between the rest and VR-EX conditions during exercise . The paired t test with Holm correction results showed significant RPE differences among all postexercise conditions .F2,22=4.2, P=.03). The paired t test with Holm correction results showed significant differences between the rest and VR-EX conditions at postsession .The POMS2 vigor-activity and fatigue-inertia results are shown in F1.26,13.87=21.14, P<.001), stability , and vitality , and a significant main effect of time on pleasure . The paired t test with Holm correction results for arousal and vitality showed significant differences between the rest and 2D-EX conditions, between the rest and VR-EX conditions after exercise, and between the 2D-EX and VR-EX conditions after exercise . The paired t test with Holm correction results for stability showed significant differences between the rest and 2D-EX conditions , and between the rest and VR-EX conditions  after exercise.The TDMS data in F1.24,13.65=28.96, P<.001). There was no significant main effect or interaction in the correct rate . This might be because the percentage of correct answers remained high, above 90% , for all participants. Conversely, the RT results verified that Stroop interference was generally observed in all the sessions of this experiment. Thus, to clarify the effect of an acute bout of exercise on a specifically defined cognitive process, we analyzed Stroop interference  by RT.First, to examine whether a general CWST tendency could be reproduced in all conditions, RT and correct rate were included in a repeated measures ANOVA using the Greenhouse-Geisser correction, with trial , condition (rest/2D-EX/VR-EX), and time (before/after) as within-participant factors. Results showed a significant main effect of trial on RT  or interaction . Then, the difference in the degree of Stroop interference between rest, 2D-EX, and VR-EX conditions was calculated using the Greenhouse-Geisser correction with condition (rest/2D-EX/VR-EX) and time (before/after) as within-participant factors. The results showed no significant main effect , RPE and vigor-activity , fatigue-inertia , arousal , pleasure , and stability . The 2D-EX condition showed no correlations between physiological and psychological parameters. sEBR was not related to any of the psychological parameters in either condition.We examined the correlation between physiological and psychological parameters in the 2D-EX and VR-EX conditions. The correlation matrix summarizing the correlation coefficients and significance results between each variable in the VR-EX and 2D-EX conditions is shown in This study investigated whether VR exergaming enhances executive function and mood. The findings showed that VR exergaming enhanced positive moods, such as vigor-activity and vitality, but did not improve executive function. These results might be explained by the increased arousal level after exercise.Using estimates of traditional predicted maximum HR , we calcThe VR-EX condition caused an increase in the POMS2 vigor-activity scale. Both the 2D-EX and VR-EX conditions increased TDMS arousal, stability, and vitality levels, but arousal and vitality levels differed between the 2 conditions. Participants performed the same-intensity exercise in both conditions, suggesting that the VR exposure might have synergistically increased arousal and vitality levels. These results replicate previous findings that VR exercise improves mood . ConversWe checked whether Stroop interference, which assesses executive function, was occurring in this experiment. The behavioral measurements revealed a shorter RT in the neutral trials than in the incongruent trials. Thus, we confirmed that Stroop interference could be induced before and after an acute bout of exercise or rest in all conditions. Based on these results, we first compared the effect of all conditions on Stroop interference and found no significant change between conditions; besides, there was no improvement in executive function in either the 2D-EX or VR-EX condition. In addition, there were no differences in executive function improvement between the VR-EX and 2D-EX conditions, despite differences in vigor, arousal, and vitality levels. These results were contrary to our hypothesis and differed from previous studies showing that 10 minutes of moderate-intensity exercise enhanced executive function . TherefoOur analysis showed an increase in arousal levels as measured by the TDMS in both the 2D-EX and VR-EX conditions, but contrary to previous studies, no association was found in the 2D-EX condition and the opposite association was found in the VR-EX condition. This phenomenon may be considered from the inverse U-shaped model of arousal levels . VR has Improved executive function from exercise is not only related to arousal level but also to pleasure level . The braFurthermore, our findings showed no improvement in executive function in either the VR-EX or 2D-EX condition. The 2D-EX condition in this study combined exercise and computer games, where participants exercised according to the target shown on the display. Therefore, our results are consistent with those of previous studies reporting that a combination of transient exercise and games did not improve executive function ,54. DualSeveral limitations of this study should be noted. First, it included only healthy adults without regular exercise habits; thus, there was insufficient interparticipant evaluation. Further validation with athletes, children, and the elderly is needed to clarify the effectiveness of VR exergaming. Second, the TDMS, which is a psychological rather than a physiological scale, was used to assess brain arousal levels. Future studies should assess physiological arousal using electroencephalograms to clarify optimal VR exergaming conditions that enhance brain function based on brain arousal level. Third, this study used FitXR, a VR exergame with a set exercise intensity; it is unclear to what extent the study results generalize to other games. Our findings suggest that games with high cognitive demands might eliminate the positive effect of exercise on executive function; however, further research is needed to determine whether games with low cognitive demands improve executive function. In addition, as mentioned earlier, we examined the transient intervention effects of VR exergaming; however, long-term intervention effects remain unclear. VR exergaming can cause negative effects, such as VR sickness , and posOur study confirms that VR exergaming improves some mood items, but did not provide evidence that it improves executive function. We found that VR exergaming with high cognitive demands may inhibit the positive effects of exercise on executive function. To propose VR exergaming that promotes exercise habituation, it is essential to further examine the combination of exercise and VR conditions that enhances both brain function and mood.


The melanocortin receptor accessory proteins (MRAP1 and MRAP2) are well‐known endocrine regulators for the trafficking and signalling of all five melanocortin receptors (MC1R–MC5R). The observation of MRAP2 on regulating several non‐melanocortin G protein‐coupled receptors (GPCRs) has been sporadically reported, whereas other endogenous GPCR partners of the MRAP protein family are largely unknown.Here, we performed single‐cell transcriptome analysis and drew a fine GPCR blueprint and MRAPs‐associated network of two major endocrine organs, the hypothalamus and adrenal gland at single‐cell resolution. We also integrated multiple bulk RNA‐seq profiles and single‐cell datasets of human and mouse tissues, and narrowed down a list of 48 GPCRs with strong endogenous co‐expression correlation with MRAPs.36 and 46 metabolic‐related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively. MRAPs exhibited protein–protein interactions and varying pharmacological properties on the surface translocation, constitutive activities and ligand‐stimulated downstream signalling of these GPCRs. Knockdown of MRAP2 expression by hypothalamic administration of adeno‐associated virus (AAV) packed shRNA stimulated body weight gain in mouse model. Co‐injection of corticotropinreleasing factor (CRF), the agonist of corticotropin releasing hormone receptor 1 (CRHR1), suppressed feeding behaviour in a MRAP2‐dependent manner.Collectively, our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad‐spectrum GPCR modulators. MRAP proteins not only serve as a vital endocrine pivot on the regulation of global GPCR activities in vivo that could explain the composite physiological phenotypes of the MRAP2 null murine model but also provide us with new insights of the phenotyping investigation of GPCR–MRAP functional complexes. We performed a single‐cell transcriptome analysis of adrenal gland and hypothalamus and drew a fine endogenous blueprint of all the emerging GPCRs and MRAP proteins.36 and 46 metabolic‐related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively.Our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad‐spectrum GPCR modulators. The melanocortin receptors (MCRs) are a subfamily of the rhodopsin class of GPCRs. Their endogenous accessory protein family named melanocortin receptor accessory protein (MRAP) contains two members in mammals, MRAP1 and MRAP2. They are single‐transmembrane proteins and function as unusual antiparallel homo‐ and heterodimers.Subsequently, numerous studies have found the wide distribution of MRAP1 and MRAP2 in several tissues beyond MC2R and MC4R expression, respectively.In this study, in order to comprehensively assess the role of the MRAP protein family on regulating the whole GPCR family, we performed a single‐cell transcriptome analysis of adrenal gland and hypothalamus, the two organs in which expression of MRAP1 and MRAP2 were enriched. We also collected and integrated multiple bulk RNA‐seq and other single‐cell datasets of human and mouse tissues and drew a fine endogenous blueprint of all the emerging GPCRs and MRAP proteins. Consistent with our in silico anticipation, in vitro studies confirmed the protein interaction of these GPCRs with MRAP proteins. Pharmacological analysis further revealed their regulatory roles on the plasma membrane translocation, constitutive activities and ligand‐stimulated intracellular signalling of GPCRs in the presence of various amount of MRAP1 and MRAP2 proteins. Moreover, knockdown of MRAP2 expression by hypothalamic administration of AAV packed shRNA stimulated body weight gain in mouse model. We selected a validated target of MRAP2, the CRHR1 for in vivo physiological evaluation. MRAP2 could inhibit the pharmacological activation of CRHR1 in vitro and hypothalamic co‐injection of CRF, the endogenous agonist of CRHR1 suppressed feeding behaviour in MRAP2 knockdown background. Overall, our data support the idea that MRAP1 and MRAP2 are functioning as broad‐spectrum GPCR modulators and may regulate the energy homeostasis of mammals in an integrated and composite manner.22.1All C57BL/6 mice were purchased from the GemPharmatech. The mice received food and water ad libitum and were maintained on a regular 12‐h diurnal cycle, and housed in disposable ventilated cages with no more than five animals per cage at 23 ± 2°C. All animal‐related experimental procedures in this study were approved by the Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine.2.2The MRAPs (MRAP1 and MRAP2) and 48 selected GPCRs were cloned from a wild‐type mouse cDNA library. The N‐terminal 3xHA and 2xFLAG tag were fused to GPCRs and MRAPs by PCR and the tagged cds sequence of receptor genes were all cloned into a pcDNA3.1(+) vector. Forty‐eight selected GPCRs were tagged with non‐fluorescent fragments of YFP F1 at C‐terminus. The C‐end of MRAP1 and MRAP2 were tagged with FLAG and non‐fluorescent fragments of YFP F2. Primer sequences are available on request. The validity of all constructs in this study was verified by DNA sequencing.Prostaglandin E2, the agonist of PTGER2 and PTGER4, was purchased from TargetMol. CL‐316,243 (the agonist of ADRB3), L‐161,982 (the antagonist of PTGER4), SR 59230A (the antagonist of ADRB3), PF‐04418948 (the antagonist of Ptger2) and Antalarmin (the antagonist of CRHR1) were purchased from R&D SYSTEMS. CRF, the agonist of CRHR1, was purchased from MCE (MedChemExpress).2.3https://www.genome.gov/27543770/gtex‐progress‐update). The overall expression patterns of the detected GPCRs were visualized by a cross‐tissue expression heatmap using R package ComplexHeatmap.We employed the online bulk RNA‐seq database GTEx to explore the expression profiles of MRAP/MRAP2 and GPCRs across 22 human tissues .The raw reads were first aligned to the mouse genome (mm10) using the 10 × Genomics CellRanger (version 3.0) package with the default parameter settings. The low‐quality cells were filtered out if they met the following criteria: (1) the detected gene number in a cell was below 300 or above 6000; (2) the total counting reads of a cell is below 500 or above 20,000; (3) the percentage of mitochondrial genes was above 25% for hypothalamus and 30% for adrenal gland; (4) the percentage of ribosomal genes was above 25% and 20% for adrenal gland. The high‐quality cells were kept for the subsequent analysis. The count normalisation, PCA dimensional reduction, cell clustering and visualisation steps were conducted with Seurat package.2.6The published scRNA‐seq datasets of mouse hypothalamus in four previous studies were obtained with the NCBI Gene Expression Omnibus (GEO) numbers: GSE87544, GSE130597, GSE74672, and GSE125065.2.72 at 37°C. Transient transfections were carried out using PEI (Polyscience) according to the manufacturer's instructions. Total plasmid concentrations of all transfections were kept consistent in each experiment by adding empty vectors.HEK293T cells were maintained in DMEM/High Glucose (Gibco) supplemented with FBS (10%) and penicillin/streptomycin (1%). Cells were cultured in a humidified atmosphere consisting of 5% CO2.8For Western blotting, proteins were extracted from mouse hypothalamus or HEK293T cells and quantified using BCA Kit (Beyotime). Blots were incubated with the following antibodies: anti‐MRAP2 (Thermo Scientific) at 1:1000 dilutions, anti‐actin (Abcam) at 1:4000 dilution, anti‐ERK1/2(Abcam) at 1:1000 dilution, anti‐pERK1/2 (Abcam) at 1:1000 dilution.n = 3).For Co‐IP experiments, HEK293T cells were transfected with indicated plasmids and lysed. Lysates were centrifuged and supernatants were incubated with rabbit anti‐HA  or rabbit anti‐Flag  at 1:5000 dilution overnight at 4°C. Next day, samples were then incubated with Protein A/G Agarose beads (Beyotime) at 4°C for 4 h. Beads were washed three times and resuspended in LDS loading buffer and boiled for 15 min. Proteins were then resolved by SDS/PAGE and detected by mouse anti‐HA  or mouse anti‐Flag  for GPCRs and MRAPs co‐IP experiments. Each IP group was repeated at least three times  at 1:5000 ratio to detect surface expression of MRAPs. After washing three times, samples were incubated with Alexa Fluor594 antibody (Abcam) at 1:5000 ratio. Finally, cells were sealed with ProLong (R) Gold Antifade containing DAPI Molecular Probes . Images were captured under 60× oil objective with Zeiss confocal microscopy (LSM880). Each YFP group was repeated at least twice, with three fields of view selected.Cells were seeded on poly‐2.10l‐lysine‐coated 12‐well plates and transfected transiently with the GPCRs (which interacted with MRAPs) and MRAP1 or MRAP2 , or empty vector only. After 24 h transfection, cells were washed three times with PBS, then fixed with 4% polyformaldehyde for 20 min. After fixation, cells were washed three times with PBS and then blocked with 5% milk in PBS for 30 min. Samples were then incubated with 1:2000 mouse anti‐HA antibody for 2 h and 1:2000 anti‐mouse‐HRP antibody  for 1.5 h at RT before the cells were washed three times with PBS. Next, TMB Substrate Solution for ELISA (Beyotime) was added and the reaction was final stopped with equal amount of 2 M sulphuric acid. The absorbance value was measured at 450 nm using a Spectramax I3 plate reader. Three replicate wells were set up for each experimental condition, and the average value was calculated at the time of measurement.Cells were cultured in poly‐2.11Cells were seeded in 24‐well plates and transfected with several plasmids, including CRE‐luciferase reporter plasmid to drive firefly luciferase by a CRE, Renilla luciferase reporter plasmid, along with responding GPCR plasmid  and either an empty vector or MRAP1/MRAP2 . After 24 h transfection, cells were incubated with varying concentrations of peptide agonist in DMEM/High Glucose with .1% bovine serum albumin. After 4 h, the medium was removed and Dual‐Glo luciferase buffer with the substrate (Promega) in DMEM/high glucose (1:1) was added. Finally, the luminescence was detected by a Spectramax I3 plate reader . Three replicate wells were set up for each experimental condition, and the average value was calculated at the time of measurement.2.12−11 to 10−6 M with the presence of 1 nM CRF; SR59230A (antagonist of ADRB3) ranged from 10−12 to 10−7 M with the presence of 1 nM CL316,243; PF‐044 18948 (antagonist of PTGER2) ranged from 10−10 to 10−5 M with the presence of 1 nM PEG2; L‐161982 (antagonist of PTGER4) ranged from 10−9 to 10−5 M with the presence of 0.1 nM PEG2 were added into plates, respectively. After 4 h incubation at 37°C, the cAMP levels were examined by the Dual‐Glo Luciferase Assay kit (Promega) according to the manufacturer's instructions. Three replicate wells were set up for each experimental condition, and the average value was calculated at the time of measurement.Cells plated in 24‐well plates were transfected the next day with the plasmids listed above. After 24 h of transfection, Antalarmin (antagonist of CRHR1) ranged from 102.132 + flux was measured by the Dual‐Glo Luciferase Assay kit (Promega). Finally, we used Firefly luciferase/Renilla luciferase to express the constitutive activity of each group of measurements.For Gs/Gi‐mediated signalling, we transfected with the indicated GPCR plasmids, MRAP1 or MRAP2, CRE‐luciferase reporter plasmid and Renilla luciferase reporter vectors into HEK293 cells per well via PEI reagent. For Gq‐mediated signalling, we transfected with a NFAT luciferase reporter plasmid instead of the plasmid encoding the CRE‐luciferase reporter to detect the calcium flux released by the activation of the Gq signalling pathway. After 24 h transfection, the luminance of cAMP levels or Ca2.14The pscAAV‐U6‐shRNA‐CMV‐EGFP‐tWPA [AAV‐ctrl] and pscAAV‐U6‐shRNA(Mrap2)‐CMV‐EGFP‐tWPA [AAV‐Mrap2] virus (Obio Technology (Shanghai) Corp., Ltd) were injected bilaterally into the hypothalamus of C57BL/6J mice. Eight weeks old mice were anaesthetised with tribromoethanol. Coordinates used for hypothalamus injection were as follows: bregma: anterior–posterior −1.5 mm; lateral ± 0.35 mm; and dorsal–ventral −5.8 mm). The AAVs were infused at a slow rate of 40 nl/min  and the injector remained in place for an additional 5 min before.2.15At least 10–14 days before the experiments, mice were continuously recorded of the amount of consumed food, weight gain and blood glucose every 2 days. Then, mice were received 0.3 μg/μl ICV injection of CRF or 1 μl PBS injection to test the inhibitory effect of the peptide on 2‐h cumulative food intake. Weight gain and blood glucose were recorded every day.2.16t‐test and one‐way analysis of variance were used. *p < .05, **p < .01, ***p < .001, ****p < .0001.Data were analysed using GraphPad Prism 6 program  and shown as mean ± SEM. To determine the statistically significant differences between groups or time points, two‐sided Student's 33.1MRAP1 and MRAP2 among the 22 human tissues and 17 mouse tissues . In addition to the classic markers to annotate the cell identities, we also found that some GPCRs  could be utilized as cell markers for labelling some hypothalamic cell types  was identified in the Mrap2 neurons of the mouse hypothalamus . We identified a total of 11 cell types in human adrenal gland scRNA‐seq data  were highly expressed in the adrenal scRNA‐seq data are in our 48 GPCR gene sets Figure , which a3.4Next, we performed co‐immunoprecipitation (co‐IP) assays to examine the ability of sorted GPCRs to form a protein complex with MRAP1 and MRAP2 proteins. GPCRs were labelled with 3xHA tag and MRAP1 or MRAP2 was labelled with 2xFlag tag. Indicated plasmids were transfected into HEK293T cells and the total plasmid amounts were kept consistent in each experiment by adding empty vectors. Next, total proteins were pulled down with a monoclonal anti‐HA antibody. The precipitated proteins from the cell lysates were sorted by Western blot with anti‐HA antibody for GPCRs or anti‐Flag antibody for MRAP1 or MRAP2. As shown in Figures To further determine the interactions in live cells and the subcellular localisation of these complexes, we performed bimolecular fluorescence complementation assays with selected GPCRs and MRAPs, as described previously.3.5Luciferase reporter assays were then performed to further verify the roles of MRAP1 and MRAP2 on the pharmacological properties of these GPCRs. In this study, we selected several GPCRs that were strongly related to energy metabolism Table  and testBased on the information in the UNIPROT database, we classified all screened GPCRs into three categories: Gs/Gi, Gq and unknown. Then, we determined their constitutive activity in the presence of MRAP1 or MRAP2 according to the classification. Among them, we measured the constitutive activity of Gs/Gi and Gq pathway both for the unknown GPCRs. The calcium signalling was assessed by NFAT luciferase activity. We found that both constitutive activity and PEG2‐inducible activity of PTGER2 and PTGER4 were suppressed by increasing expression of MRAP1 or MRAP2 Figures , which sNext, we measured the constitutive Gs(cAMP) or Gq(Ca2+) activities of selected GPCRs in the presence of various doses of MRAP1 or MRAP2 Like MC4R, most of the identified GPCR targets exhibited basal constitutive activity in the absence of agonists. MRAP proteins dramatically altered the basal intracellular cAMP or Ca2+ levels of most GPCRs Figures  and S7. 3.6Given that the MRAP proteins interacted with most GPCRs, we next investigated the mechanism by which MRAP proteins regulated these GPCRs. MC4R activation, which is primarily regulated by MRAP2, leads to cAMP synthesis and activates the downstream signalling pathway and phosphorylates extracellular signal‐regulated kinase 1 and 2 (ERK1/2).Since the presence of MRAP proteins affected the cell surface translocation of MCRs, we then tested whether MRAP proteins could modulate the post‐translational cell surface trafficking of the receptor. Next, we transfected HEK293T cells with 3HA‐tagged GPCRs with either MRAP1, MRAP2 or a blank vector. We then measured the cell‐surface expression of GPCR in non‐permeabilised fixed‐cells with enzyme‐linked immunosorbent assay (ELISA). As shown in Figures 3.7MRAP2 deletion or loss‐of‐function mutation resulted in obesity syndrome in mice and human.4The MC4R, regulated by MRAP2, shows very extensive dimeric binding of numerous GPCR targets in the central nervous system.Activation of the hypothalamic–pituitary–adrenocortical (HPA) axis can respond to acute threats to homeostasis or health state. An increase in circulating glucocorticoids mobilises energy and promotes some behavioural or physiological responses to stressors.Here, we assessed the correlation coefficient of MRAP proteins and GPCRs in co‐expressing cells of the hypothalamus and adrenal gland, the two major endocrine organs for exerting the physiological functions of MRAP proteins Figure . We alsoThe following co‐IP results showed obvious interactions between most sorted GPCRs and MRAPs  Figure . All of These findings spurred us to further assess the influence of MRAP proteins on GPCR signalling. We examined the pharmacological activity of four GPCRs interacted with MRAP proteins and related to metabolism: ADRB3, CRHR1, PTGER2 and PTGER4 with or without MRAPs in different concentrations of agonists or antagonists Figure . MRAP1 aSeveral prior studies have noted the vital role of MRAP2 on regulating non‐melanocortin GPCRs. For example, Srisai et al.Further in vivo evidence from animal studies, along with Genome Wide Association Studies (GWAS), pointed out that most of the identified novel GPCR partners interacting with MRAP1 or MRAP2 may have relevance to energy homeostasis and deficiency of which could lead to metabolic disorders and other neuronal related diseases Table . InteresIt is undeniable that some GPCRs are low expressed, but GPCRs act in a cascade effect and cells do not require much GPCR expression to function. In addition, at the single cell level only 3K–4K genes are generally detected, so many GPCRs are not visible at the mRNA level and may be overlooked. It must be stated that low GPCR expression may also give rise to important functions and these invisible GPCRs may functionally related to MRAP2. In summary, MRAP1 and MRAP2 are presented and redefined as broad‐spectrum GPCR modulators and act as vital physiological links to a large group of GPCRs in regulating energy homeostasis. Our findings could help us better understand the composite obese phenotype resulting from MRAP2 deficiency in vivo. The universal binding of MRAP2 to various GPCRs provides us new strategies for the development of novel transmembrane allosteric modulators for several pharmaceutically important GPCR targets.The authors declare that they have no competing interests.Supporting InformationClick here for additional data file.Supporting InformationClick here for additional data file.


Toughness of epoxies is commonly improved by adding thermoplasticphases, which is achieved through dissolution and phase separationat the microscale. However, little is known about the synergisticeffects of toughening phases on multiple scales. Therefore, here,we study the toughening of epoxies with layered poly(ether imide)(PEI) structures at the meso- to macroscale combined with gradientmorphologies at the microscale originating from reaction-induced phaseseparation. Characteristic features of the gradient morphology werecontrolled by the curing temperature (120–200 °C), whilethe layered macro structure originates from facile scaffold manufacturingtechniques with varying poly(ether imide) layer thicknesses (50–120μm). The fracture toughness of the modified epoxy system isinvestigated as a function of varying cure temperature (120–200°C) and PEI film thickness (50–120 μm). Interestingly,the result shows that the fracture toughness of modified epoxy wasmainly controlled by the macroscopic feature, being the final PEIlayer thickness, i.e., film thickness remaining after partial dissolutionand curing. Remarkably, as the PEI layer thickness exceeds the plasticzone around the crack tip, around 62 μm, the fracture toughnessof the dual scale morphology exceeds the property of bulk PEI in additionto a 3 times increase in the property of pure epoxy. On the otherhand, when the final PEI thickness was smaller than 62 μm, thefracture toughness of the modified epoxy was lower than pure PEI butstill higher than pure epoxy (1.5–2 times) and “bulktoughened” system with the same volume percentage, which indicatesthe governing mechanism relating to microscale interphase morphology.Interestingly, decreasing the gradient microscale interphase morphologycan be used to trigger an alternative failure mode with a higher cracktortuosity. By combining facile scaffold assemblies with reaction-inducedphase separation, dual-scale morphologies can be tailored over a widerange, leading to intricate control of fracture mechanisms with ahybrid material exceeding the toughness of the tougher phase. Unfortunately, for some tougheners, the additionof the second phase into the epoxy system also reduces overall modulusand limits the end-use temperature of the system.5 Moreover,the viscosity of the resin system may also increase significantlyby bulk resin modification, primarily through thermoplastics, whichmakes them unsuitable for liquid composite molding processes.6Epoxy-based compositesare subjected to high static and dynamicloadings in engineering applications, which require higher resistanceto fracture. In contrast, epoxies with high cross-linking densitiesare inherently brittle and typically have a low fracture toughness.However, different approaches are known to increase their fracturetoughness.8 Combining theseeffects results in hierarchically toughened structures. The firstand most relevant approach for toughening on a macro scale is theintroduction of ductile interlayers into a brittle material, whichleads to toughening through the inhomogeneity effect.10 The material inhomogeneity effect is based on spatial variationsin material properties significantly influencing the crack drivingforce, i.e., a material inhomogeneity can hinder or promote crackpropagation. The ductile interlayers act as crack arresters, so thatthe fracture toughness of the base material strongly increases. Ifthe ductile interlayers are thin enough, the loss in strength is almostnegligible.11 For instance, Sistaniniaet al. focused on designing new fracture resistance material by utilizationof the material based on the yield stress inhomogeneity effect.12 They used ductile interlayers (low-strengthsteel) to toughen the high-strength steel matrix and reported a criterionof optimum architectural parameters, i.e., optimum interlayer spacing.The multilayer specimen’s fracture toughness was at a similarvalue to the sample having a single interlayer in the case of interlayerspacing larger than the process zone around the crack tip. Likewise,Zechner and Kolednik used aluminum sheets as multilayers with a polymeradhesive.13 Structures in different configurations are tested. The resultsshowed that the crack arrester configuration exhibits a tremendouslyimproved crack growth resistance (73.7 kJ/m2) and fractureinitiation toughness compared to the single sheet configuration (33.6kJ/m2), which was attributed to the so-called “materialinhomogeneity effect”.13 Severalother studies introducing soft interlayers into brittle material showedincreased fracture toughness due to the inhomogeneity effect.15 All of these studies highlighted the importance of material inhomogeneityat specific scales in improving the fracture toughness of brittlematerial. This concept is also adopted in this current study to toughenan epoxy system  by using multilayers of poly(etherimide) , leading to the material inhomogeneityeffect .In contrast to bulk modification, severalother approaches andeffects to toughen a brittle material have been observed on the macro-,the micro-, and/or the nanoscale.19 For instance, Harismendy et al.,reported the formation of a particulate morphology by adding 10 wt% PEI into the epoxy which resulted in a 2-fold increase in fracturetoughness due to crack path deflection mechanism.16 Likewise, as reported in our previous study,20 in the case of using a thermoplastic film asthe ductile interlayer, liquid reactive thermoset monomers diffuseinto the glassy thermoplastic and partially swell or dissolve it,which results in the diffusion of TP polymeric chains into the epoxyresin. The mutual diffusion of the components creates a concentrationgradient in the interfacial region.21 Theproceeding cure reaction between the comonomers induces a phase separationleading to a gradient morphology in the interfacial zone.22 The gradient interphase shows different morphologiesthroughout its thickness depending on the concentration of epoxy andPEI. This granular morphology can impart toughening effects similarto those of micro- and nanosized dispersed particles, as found inbulk toughening of thermosetting systems. These “inclusions”can induce various energy dissipation mechanisms, thereby limitingthe onset and propagation of cracks by different toughening mechanisms,i.e., crack deflection, crack pinning, or debonding of particles.24 The cure profile strongly affects the thickness of the interphaseand its morphology.20Another potentialtoughening effect occurs at one scale lower,at the microscale. Several researchers have reported the microtougheningof epoxy using a thermoplastic (PEI).The aim ofthis study is to understand the effect of micro- andmacrotoughening approaches on the fracture toughness of an epoxy systemby using thermoplastic PEI multilayers. Thereby, the following researchquestions are addressed: (i) what are the respective effects of macrotoughening and microtoughening  on the fracture toughness of epoxy? (ii) Is there any synergeticeffect between these two toughening mechanisms? From the morphologicalpoint of view, it is not known whether a larger scale heterogeneityin the submillimeter range, by having thermoplastic multilayers, wouldpossibly be more effective. Therefore, the first research questionis addressed by toughening the epoxy resin using multilayers of PEIwith varying thicknesses (50–120 μm) to tune the macrotougheningphenomenon. The second research question is investigated by developingdiffusion-controlled interphase gradients (micron-sized morphology)between the aforementioned multilayers at the epoxy/PEI interfaceas a function of different cure temperatures (120–200 °C)and determining the resultant fracture toughness of the system. We,therefore, present a method to create dual-scale morphologies by (a)assembling multilayer scaffolds of thermoplastic tougheners with architectedporosity (giving rise to the morphology in the submillimeter range)and (b) creating diffusion-controlled interphases (micron-sized morphology)along with the reaction-induced phase separation after infiltrationwith an epoxy system. This allowed us to discriminate the effect ofthe thickness of the thermoplastic layer, cure temperature, interphasethickness, and morphological features on the fracture toughness ofthe resultant hybrid material.22.1n ≈90, Tg = 217 °C), with differentthicknesses , provided by SABIC, SaudiArabia, were used as the tough phase. The thermoset resin was preparedas a blend of M--N,N-bisaniline , and bisphenol-Fepoxy resin monomer . 3,3′-sulfonyldianiline was used as a curing agent for epoxy. Huntsman,Switzerland, supplied all these chemicals.Thin films of PEI  was then incorporatedinto 50 g of epoxy resin. The prepared mixture was stirred with aspeedmixer at 1200 rpm for 4 min. The epoxy resin was degassed byusing vacuum for 5 min to remove the entrapped air bubbles.2.2.2PEI scaffoldswere prepared by stacking the PEI films periodically in a 1:2 ratioof equal thickness as “PEI layer”, “spacer”,“spacer”, “PEI layer”, “spacer”,“spacer”, “PEI layer”, “spacer”,“spacer”, and so on 1 to obta2.2.325 The timeand temperature during the curing stage  cure temperatures of 120,140, 160, 180, and 200 °C were selected to attain at least 70%degree of cure at the first dwell. The postcure time and temperaturewere selected to attain full cure of the sample.In this study, the PEI-toughenedepoxy samples were cured using a two-dwell cure cycle approach alreadyreported in the literature.2.2.426 The dimensions of the SENB sample were as follows:length = 26.4 mm, width = 6 mm, and thickness = 3 mm.The fracturetoughness of the specimens was determined by single-edge notched bending(SENB) testing according to the ASTM D5045-99 standard.a0) with the help of a saw blade of 0.12 mm thickness. Then, the notchwas filled with diamond paste ; a razor blade wasinserted in the notch, and a natural crack (Δa) was obtained by sliding the razor blade (see As shownin lade see 2.a0 + Δa) was ±1.7mm. All ofthe tests were conducted on a Zwick/Roell tensile test machine witha 1 kN load cell and a 10 mm/min testing speed. The device was setup according to the ASTM standard D5045-99.26 The resulting load–displacement curves were then translatedto KIc and GIc values. The plastic zone around the crack tip in our system wasestimated to be 62 μm by the Irwin model27 under the plane strain condition (see the Supporting Information). Therefore, linear elastic fracturemechanics (LEFM) was applied to the system because the radius of theplastic zone was smaller compared to the crack and ligament length.27The total length of the notch and natural crack was used to etch the polishedsamples following the procedure explained elsewhere.20 The etched samples were coated with gold using a sputtercoater and then analyzed using SEM. For fractography analysis, thecross-section of fractured samples obtained after SENB testing wasalso coated with gold and analyzed using SEM.The interphaseand morphology of the cured PEI toughened epoxy samples were studiedby using field emission scanning electron microscopy (SEM)  and confocal laser scanning microscopy (CLSM) . For morphological analysis, samples were embedded in afast-cure epoxy resin, followed by grinding and polishing. 33.120 The size of these epoxy droplets was observed to decrease graduallytoward the pure PEI region due to the increase in PEI content. Similarresults were also obtained for the samples cured at 140, 180, and200 °C.The interphaseformation between epoxy and PEI at different cure temperatures wasanalyzed by using CLSM and SEM. An example of the multilayer sampleconsisting of a PEI layer of 90 μm cured at 160 °C (1stdwell temperature) is shown in Most of the samples showed a distinct gradientmorphology. However,two cure conditions showed different behaviors: (1) samples curedat 120 °C and (2) samples cured at 200 °C with a low PEIlayer thickness. For the first case, no visible gradient morphologywas observed for samples cured at 120 °C at the given resolutionfor all of the studied PEI layer thicknesses. For example, the SEMimage of a sample with 60 μm PEI film cured at 120 °C isshown in On the other hand, the maximum interphase thicknessfor a 50 μmPEI layer seems to be found at 200 °C, which, however, was linkedto the complete dissolution of the monolithic PEI layer and difficultyin analyzing the thickness of a single interphase, which would behalf of the measured value. Hence, in the case of the 50 μmPEI layer, the maximum interphase thickness was evident at a curetemperature of 180 °C. This behavior shows that the criticalcure temperature required for achieving maximum interphase thicknessshifted toward a higher value as a function of decreasing PEI thickness.Furthermore, this graph indicates that the higher the PEI layer thickness,the higher the interphase thickness at any cure temperature. For instance,the interphase thickness is 72 μm for sample 12P14 with a PEIlayer thickness of 120 μm, while for the same cure conditions,with a PEI layer thickness of 50 μm (sample 5P14), it is 33.4μm.3.220 are plotted asa function of position along the interphase for samples cured at differenttemperatures .The morphologygradient consists of epoxy-rich droplets in a continuous PEI-richphase. The epoxy droplet size and frequency, calculated from blobdetection with OpenCV,eratures 6. At 120eratures 6a, a nareratures 6b, indic3.3For SENB of multilayersystems, the precrack tip location in the tough PEI or brittle EPphase influences fracture behavior significantly. In S3. It is witnessed that in the case of 6P12,crack initiation happened in the epoxy phase (step 1A) as the cracktip was in the epoxy phase, which caused an increase in the load untilstep 1B. After that, a sharp decrease in load is found as the crackpasses through interface 1 . The solidline in KIc value calculated accordingto the rule of mixtures16epoxy = 2/3 and ϕPEI = 1/3 represent the volume fractions of epoxy and PEI,respectively. The rule of mixture represents the bulk resin modification,and therefore, The effect of cure temperature and PEI film thicknesson the fracturetoughness and critical energy release rate of samples is shown in KIc) and energyrelease rate (GIc) displayed a decreasingtrend with the increase in cure temperature. The increase in the planestrain fracture toughness, KIc, and energyrelease rate, GIc, compared to pure PEI,particularly at the lower cure temperatures (120–140 °C),strongly suggests the synergistic benefit of multilayer architectureand morphologies present at the interphase.Both fracture toughness . At 140 °C, the remainingPEI layer thickness was similar to the plastic zone size in the caseof the 90 and 120 μm initial PEI layer, while the remainingPEI layer thickness for the 50 and 60 μm initial PEI layer wasless than the plastic zone. At higher cure temperatures, the remainingPEI layer thickness was well below the plastic zone size for all ofthe initial PEI thicknesses.The value of 4The enhancement in fracturetoughness of epoxy toughened with PEImultilayers is observed on two levels, i.e., (1) macroscale and (2)microscale.4.128 found an increase in strain energy when a crack grows from a brittleto a ductile material, called the antishielding effect. On the otherhand, the strain energy is diminished when a crack grows from theductile to the brittle phase. This so-called shielding effect canstop the crack from growing entirely, leading to crack arrest at theinterface.The macro-scale tougheningof epoxy comes from introducing tough and ductile PEI layers intothe epoxy, which creates the inhomogeneity effect . Several studiestcritical) is equal to the plastic zone size.29 At a low cure temperature . Therefore,this result shows that the remaining PEI thickness  plays a dominant rolein defining the macrotoughening phenomenon, where the critical valuewas found to be in the range of the plastic zone of the PEI, about∼60 μm.It is reported that the maximum inhomogeneity effectis achieved when the thickness of the interlayer . There are three regions of interestin this morphology: (1) PEI inclusions in the epoxy-rich phase, (2)the interphase region (with or without the gradient morphology dependingupon the cure temperature), and (3) the PEI-rich phase with epoxyinclusions. The epoxy inclusions in the PEI-rich phase were not discussedin the following sections due to their negligible effect on the fracturebehavior of samples. Therefore, the microscale toughening of epoxywas explained based on two remaining regions: (i) gradient morphologyin the interphase region and (ii) PEI inclusions in pure epoxy.As already discussed,liquid reactive thermoset monomers diffuse into the glassy thermoplasticand partially dissolve it. This mutual diffusion of the componentscreates a gradient morphology in the interfacial region and cured at 200 °C showed a complete dissolution of the PEIlayer  at a cure temperature of 120 °C 6a, suggeeratures6b, whichEIlayer 4b.In thrulesee 10a,whicThe size of the epoxy particles in the gradientinterphase governstheir connectivity with the thermoplastic phase. 32 A crack generally propagates in the direction of the least resistance.Therefore, at lower temperatures, the crack prefers to propagate aroundthe interface instead of propagating through the interface due tothe weak adhesion , the interphase typically shows the presenceof only bigger particles  calculated by Griffith’stheory.cles see 4a, which4.2.234 The fracture surfaces of the epoxy-rich phase with PEI inclusionsshow different toughening mechanisms at different cure temperatures.The epoxy-rich phase shows a ductile deformation (3D pattern) in thecase of low cure temperature , strongly suggests the dominanceof the macrotoughening phenomenon coming from multilayer architecture(To identify the main controlling parameter (cure temperature orPEI film thickness) for modifying the fracture toughness of epoxy,plane strain fracture toughness, ring see 14. This itecture14. This36 Furthermore, by decreasing the final PEI film thickness, the mechanicalproperties of PEI-toughened epoxy approach the theoretical valuesof KIc and GIc .On the other hand, when the final PEI thicknessis smaller than62 μm, the fracture toughness of modified epoxy is lower thanpure PEI but still higher than pure epoxy and “bulk toughened”systems with the same volume percentage, which shows that the governingmechanism, in this case, is microtoughening. This mechanism mainlycomes from the presence of bigger epoxy particles in gradient morphology,which plays a significant role during fracture  and gives higher crack resistance. Moreover, with a decreasein the final PEI thickness, the spacing between the two PEI layersincreases, eventually resulting in lower fracture toughness. Likewise,Kolednik et al. and several other studies found that the lower thespacing between two interlayers, the more the fracture toughness forthe given thickness. and GIc 14 obtain and GIc 1. In litKIc) as a function of processing parameters (cure temperature), morphology(interphase thickness), and architectural configuration (thicknessof the PEI film). This graph shows that a lower interphase thicknessresults in better fracture toughness of the epoxy system and PEI film thickness (50–120 μm). PEI scaffolds wereprepared by stacking the PEI films periodically in a 1:2 ratio ofequal thickness, which resulted in scaffolds with 33 vol % of PEIand 66 vol % of epoxy. This study cured the PEI-toughened epoxy samplesusing a two-dwell cure cycle.1/2) at higher final PEI film thickness (95 μm).However, changing the initial PEI film thickness or cure temperaturecan control this final PEI film thickness. Compared to pure PEI, thehigher fracture toughness of toughened epoxy, particularly at thehigher final PEI film thickness (larger than 62 μm), stronglysuggested the occurrence of both macro- and microtoughening. However,the dominant mechanism is macrotoughening coming from the multilayerarchitecture. This dominance mainly comes from the effective inhomogeneityeffect due to the critical final PEI layer thickness. On the otherhand, the fracture toughness of modified epoxy was lower than purePEI but still higher than pure epoxy and bulk-modified with a similarvolume fraction when the final PEI thickness was smaller than 62 μm,which showed that the only governing mechanism was microtoughening.This mechanism mainly comes from bigger epoxy particles in gradientmorphology, which plays a significant role during fracture  and gives higher crack resistance. This resultsshowthat for high-temperature processing, the initial PEI film thicknesshas to be selected such that the remaining film thickness allows usto obtain the synergy between micro- and macrotoughening.The result shows that the fracturetoughness was mainly controlledby the final PEI thickness, i.e., higher fracture toughness (4.53MPa mKIc and GIc obtained from the mixture rule.This study provides an understanding of the hierarchical tougheningof epoxy using a multilayer system which can further be explored inthe field of fiber-reinforced composites for aerospace applications.Moreover,with the decrease in the final PEI thickness, the spacingbetween the two PEI layers increases, eventually resulting in lowerfracture toughness. However, in the current study, the influence ofinterlayer spacing cannot be investigated because of the fixed EP/PEIvolume ratio. Therefore, in the future, to effectively understandthe influence of interlayer spacing on the fracture toughness of epoxy,further research needs to be performed without fixing the volumetricratio between epoxy and PEI. Furthermore, by decreasing the finalPEI film thickness, the mechanical properties of PEI-toughened epoxyapproach the theoretical values of


The poor prognosis of most cases of advanced cholangiocarcinoma (CCA) constitutes a severe problem in modern oncology, which is aggravated by the fact that the incidence of this liver cancer is increasing worldwide and is often diagnosed late, when surgical removal is not feasible. The difficulty of dealing with this deadly tumor is augmented by the heterogeneity of CCA subtypes and the complexity of mechanisms involved in enhanced proliferation, apoptosis avoidance, chemoresistance, invasiveness, and metastasis that characterize CCA. Among the regulatory processes implicated in developing these malignant traits, the Wnt/β-catenin pathway plays a pivotal role. Alteration of β-catenin expression and subcellular localization has been associated with worse outcomes in some CCA subtypes. This heterogeneity, which also affects cellular and in vivo models commonly used to study CCA biology and anticancer drug development, must be taken into account for CCA investigation to more accurately extrapolate basic laboratory research to the clinical situation. A better understanding of the altered Wnt/β-catenin pathway in relationship with the heterogeneous forms of CCA is mandatory for developing novel diagnostic tools and therapeutic strategies for patients suffering from this lethal disease. Cholangiocarcinoma (CCA) is an extremely heterogeneous malignancy arising in cholangiocytes, i.e., the epithelial cells that line the bile ducts . CCA conOpisthorchis viverrini, advanced age, and alcohol consumption have been described as risk factors for CCA development [A recent meta-analysis revealed that bile duct cysts and stones, cirrhosis, and viral hepatitis B and C, but not hypertension and obesity, are risk factors for all types of CCA ; howeverelopment .An important proportion of CCA cases arises in patients with non-diagnosed underlying diseases, but it is accepted that chronic cholestasis/inflammation leads to cholangiocarcinogenesis by inducing DNA damage, apoptosis evasion, promotion of cell proliferation, and neoangiogenesis . Bile acAccording to their transcriptomic profiles, iCCAs are divided into the “inflammation” and the “proliferation” subtypes ; the firThe Wnt/β-catenin signaling pathway is not the most important oncogenic pathway in CCA, but it certainly plays a role and contributes to the heterogeneity of this tumor. β-catenin is a component of adherens junctions, cell–cell adhesion complexes essential for adhesion and anchoring of the actin cytoskeleton , localizWnt ligands are cysteine-rich glycoproteins of 350–400 amino acids  secreted by different cell types that can act in an autocrine or paracrine manner .Wnt ligands can also trigger a β-catenin–independent signaling pathway, the non-canonical planar cell polarity (PCP) Wnt pathway, through activation of the small GTPases Rho and Rac, which induce cytoskeletal rearrangements and polarization of epithelial cells ,23,24. LMorphologically, CCA is characterized by an abundant desmoplastic stroma surrounding the tumor, with dense connective tissue, disorganized blood vessels, and inflammatory macrophages, which are considered an important source of Wnt ligands in these tumors. Activation of the Wnt/β-catenin signaling pathway is frequent in CCA and is associated with tumor cell proliferation . AccordiAs mentioned, it has been described that in CCA (similar to other tumors), Wnt/β-catenin activity is pathologically activated . MutatioCTNNB1 gene), which increase protein stability. This type of mutation has been found in HCC with a frequency of ≈19% [CTNNB1 is the most recurrently mutated gene, with a frequency of 50–90%, which leads to consider CTNNB1 as a driving proto-oncogene [CTNNB1 mutations are usually absent in CCA. Some studies have not found CTNNB1 mutations in CCA specimens [CTNNB1 gene in CCA is <2% , with a frequency of 14.3% of cases [APC gene lead to a less effective degradation complex, which causes β-catenin accumulation in the cytoplasm and nuclei, resulting in an aberrantly activated pathway.One of the Wnt pathway tumor suppressor genes with the most mutations in patients with CCA is  disease . The COS samples . Other sr tumors ,46. NGS of cases . Epigeneof cases  includinAXIN1 gene that generate inactive proteins. AXIN1 has a mutation frequency of 1–2% in patients with CCA, according to data from CBioPortal and COSMIC  were identified in three out of four patients [CTNNB1 were identified in the same tumors. Axin2, like axin1, acts as a scaffold to help assemble the β-catenin destruction complex; both proteins show high similarity and are considered functionally equivalent. However, only axin2, but not axin1, is a transcriptional target of β-catenin–dependent Wnt signaling. Thus, axin2 protein levels could be a key negative feedback mechanism for the regulation of Wnt/β-catenin signaling in cells [AXIN2 gene in patients with CCA is also very low  (AXIN2 gene only in two of these patients (3%), both female, one of each type of tumor, and alterations were associated with tumor mutational burden [AXIN2 have been associated with poor prognosis in other early-stage solid tumors [DVL2, DVL3, FZD10, WNT10B, and WNT8B  . The anal burden . Geneticd tumors . Other giew, see ).The Wnt/β-catenin signaling pathway has multiple nodes of interaction with other signaling cascades and is regulated in a complex cellular environment. It has been described that the Wnt/β-catenin pathway is highly active in CCA because this tumor presents a high Wnt state, which is maintained by inflammatory macrophages in the surrounding stroma . In thisRNF43 as a tumor suppressor gene [In addition to high levels of Wnt factors, other mechanisms may increase the activity of the Wnt/β-catenin pathway in CCA. For example, it is well known that the transcription factor SOX17 is downregulated in CCA due to hypermethylation of its promoter . SOX17 psor gene .The expression levels of several microRNAs targeting elements of the Wnt/β-catenin pathway are altered in CCA compared with paired non-tumor tissue . For exaThe expression of β-catenin is higher in cholangiocytes than in hepatocytes. Due to the association of β-catenin with E-cadherin-based adherens junctions, in healthy liver, β-catenin is localized at the plasma membrane and, to a much lesser extent, also in the cytoplasm of both types of liver cells A. ComparSeveral studies have investigated β-catenin expression and localization in CCA by immunohistochemistry , since tThe reduction in the presence of β-catenin at the plasma membrane usually parallels a decrease in the expression levels of other intercellular junction markers, such as E-cadherin or α-catenin ,69. β-caThere is controversy regarding the existence of differences in the frequency of nuclear staining of β-catenin depending on the CCA type. One study reported higher nuclear levels of β-catenin in pCCA than in iCCA , whereasIn patients with cHCC-CCA, a relationship between reduced β-catenin membranous expression and tumor progression and metastasis, together with higher differentiation of HCC components, has been found . In thatCCA-derived cell lines have been helpful in investigating the association of β-catenin activation and tumor cell aggressiveness and in further understanding the functioning of this signaling pathway. Higher protein expression levels of β-catenin were found in the metastasizing cell line OZ compared with the moderately invasive cell line HuCCT1, but β-catenin expression did not account for cell invasiveness in the OZ cell line . The anaβ-catenin has been linked to the development of a more malignant phenotype in initiated cholangiocytes, since exposing these cells to exosomes isolated from CCA cells resulted in the nuclear expression of β-catenin . The tumConsidering the importance of the Wnt pathway in the tumorigenesis of several types of cancer, a great effort has been made to find compounds able to inhibit the activity of this pathway. In this sense, many patents in the area of Wnt modulation have been filed, and some small molecules or antibodies have been included in clinical trials to evaluate their usefulness as Wnt modulators (for a review see ). The moOne strategy to reduce Wnt/β-catenin signaling is the inhibition of porcupine, a membrane-bound O-acyltransferase that catalyzes the acylation of Wnt proteins . This poDickkopf-1 (DKK1) is upregulated in many types of cancer, and its inhibition has been associated with decreased tumor proliferation, migration, and invasion in preclinical trials . DKK1 haAnother possibility for inhibiting this pathway is to block transcription of β-catenin target genes by preventing the nuclear interaction between β-catenin and its transcription factors TCF/LEF. Several known drugs and natural molecules have this effect, such as vitamin D and retinoic acid ,91 or thAnother compound with a similar mechanism of action is BC2059 (tegavivint), a Wnt/β-catenin pathway inhibitor that disrupts the binding of β-catenin to Transducin β-like protein 1 (TBL1), a key player in enhancing the canonical Wnt signaling pathway via direct binding to β-catenin and recruiting it to the promoter of Wnt target genes, thereby facilitating β-catenin destruction . In vitrIn addition, the non-steroidal anti-inflammatory drugs sulindac and aspirin have proved to promote the degradation of β-catenin . The effDifferent preclinical studies have shown that docosahexaenoic acid (DHA), an omega 3 polyunsaturated fatty acid, induces GSK3β dephosphorylation in human CCA cells . This acMYC and CCND1 [miRNAs have been identified that play a role in regulating the activity of the Wnt/β-catenin pathway. Specifically, a preclinical study showed that inhibition of miRNA-26a prevented β-catenin activation and transcription of its target genes and slowed the rate of tumor growth, an effect caused by this microRNA blocking the GSK3β mRNA . Similarnd CCND1 .In vitro and in vivo assays have shown that restoration of SOX17, whose expression is markedly reduced in CCA compared to healthy cholangiocytes, decreases the activity of the Wnt/β-catenin pathway, reducing tumor proliferation and progression . ExperimIt is important to note that although it was a priori thought that blocking the Wnt pathway would have no side effects in normal cells because this pathway is only activated in tumor cells, some side effects have been described upon using different inhibitory strategies. For example, the inhibition of the Wnt/β-catenin pathway disrupts intestinal homeostasis and induces severe loss of crypt-villi structure in mouse intestine . SimilarSeveral therapeutic agents specifically targeting the Wnt/β-catenin pathway have entered clinical trials, but none have yet been approved. Some trials in which Wnt pathway inhibitory agents have been administered in combination with chemotherapeutic drugs have obtained better results than monotherapy, without an increase of adverse effects . Since tThe poor prognosis of most cases of advanced CCA constitutes a severe problem in modern oncology. The difficulty of dealing with this limitation is augmented by the heterogeneity of CCA subtypes and the complexity of mechanisms involved in enhanced proliferation, avoidance of apoptosis activation, chemoresistance, invasiveness, and metastasis. Among the regulatory processes involved in developing these malignant characteristics, the Wnt/β-catenin pathway plays a pivotal role. Alteration of β-catenin expression and subcellular localization has been associated with worse outcomes in some CCA subtypes. This heterogeneity, which also affects cellular and in vivo models of CCA, must be taken into account for CCA investigation. Recommendations on the minimal criteria for preclinical models of CCA to provide a uniform approach have been recently published  to more