The purpose of our research was to determine the prognostic impact and clinicopathological feature of c-MYC and β-catenin overexpression in colorectal cancer (CRC) patients.Using immunohistochemistry (IHC), we measured the c-MYC and β-catenin expression in 367 consecutive CRC patients retrospectively (cohort 1). Also, c-MYC expression was measured by mRNA in situ hybridization. Moreover, to analyze regional heterogeneity, three sites of CRC including the primary, distant and lymph node metastasis were evaluated in 176 advanced CRC patients (cohort 2).P = 0.011, P = 0.012 and P = 0.033, respectively). Moreover, co-expression of c-MYC and ß-catenin was significantly correlated with longer survival by univariate (P = 0.012) and multivariate (P = 0.048) studies. Overexpression of c-MYC protein was associated with mRNA overexpression  and nuclear ß-catenin expression . Expression of c-MYC and ß-catenin was heterogeneous depending on location in advanced CRC patients (cohort 2). Nevertheless, both c-MYC and ß-catenin expression in primary cancer were significantly correlated with improved survival in univariate (P = 0.001) and multivariate (P = 0.002) analyses. c-MYC and ß-catenin expression of lymph node or distant metastatic tumor was not significantly correlated with patients’ prognosis (P > 0.05).In cohort 1, c-MYC protein and mRNA overexpression and ß-catenin nuclear expression were found in 201 (54.8 %), 241 (65.7 %) and 221 (60.2 %) of 367 patients, respectively, each of which was associated with improved prognosis (Co-expression of c-MYC and ß-catenin was independently correlated with favorable prognosis in CRC patient. We concluded that the expression of c-MYC and ß-catenin might be useful predicting indicator of CRC patient’s prognosis.The online version of this article (doi:10.1186/s12885-016-2770-7) contains supplementary material, which is available to authorized users. The t tumors , 4 and mt tumors , 6. Dereogenesis .c-MYC gene amplification, translocation, and alteration of regulatory molecules are major causes of c-MYC protein overexpression [c-MYC amplification and overexpression was showed in approximately 10 and 70 % in CRC, respectively [APC gene mutation, a key driver of adenoma-carcinoma transition, often leads to altered ß-catenin regulation via the well-studied Wnt signaling pathway [c-MYC and cyclin D1 [pression , 9. Prevectively ; in CRC, pathway –14. Reguyclin D1 , 16. Recyclin D1 , 18. It Nevertheless, there were a few researches that reported clinicopathological impact of c-MYC and ß-catenin status in CRC. Their prognostic value for CRC patients remains debatable. A recent study reported that c-MYC protein overexpression obtained by immunohistochemistry (IHC) was significantly correlated with better survival of CRC patients . In contKRAS, EGFR, and BRAF mutation, thus tumor heterogeneity may explain this discrepancy between molecular alteration and responses of targeted therapy [Recently, the systemic chemotherapy in CRC has made a remarkable development, and targeted therapy has been used to increase survival in advanced CRC patients . However therapy –25. TherThe aim of our research was to evaluate the clinical implication of c-MYC and ß-catenin in CRC and evaluate their heterogeneity in primary and distant metastatic tumors. We also analyzed the association between c-MYC and ß-catenin status.A total of 543 CRC cases of this study had been collected in our previous study . To inveTissue microarray (TMA) was constructed with representative lesions of the donor formalin-fixed paraffin-embedded (FFPE) CRC tissues as previously described .). ß-catenin IHC used a commercially available antibody against ß-catenin . The staining process was performed using an automated immunostainer , according to the manufacturer’s recommendations. Normal colonic mucosa cells were considered as internal negative controls. Normal mucosa was negative for c-MYC nuclear immunostaining. ß-catenin was negative in inflammatory cells, but expressed in colonic epithelium in three patterns: membrane, cytoplasm, and nucleus. We only found ß-catenin nuclear expression in malignant cells. For statistical analysis, c-MYC and ß-catenin immunostaining were regarded as positive when they were expressed in more than 10 % of neoplastic nucleus in any intensity  was used according to the manufacturer’s protocols. The experimental data was interpreted according to the manual in the RNAscope FFPE assay kit: no staining or less than 1 dot/cell at 40× objective view (score of 0); staining in 1–3 dots/cell visible at 20–40× objective view (score of 1); staining in 4–10 dots/cell with no or very few dot clusters visible at 20–40× objective view (score of 2); staining in >10 dots/cell with less than 10 % of positive cells having dot clusters visible at 20× objective view (score of 3); staining in >10 dots/cell with more than 10 % of positive cells having dot clusters visible at 20× objective view (score of 4). A score of 2–4 indicates c-MYC mRNA overexpression  and dapB  were used for positive and negative controls. Tissues were regarded as appropriate when the UBC mRNA signals were visible without difficulty at 10× magnification and the dapB signal was not visible.For the detection of Microsatellite instability (MSI) examination using fragmentation assay of ABI-3130xl with five microsatellite markers  were analyzed according to the instruction demonstrated previously . MSI exaP < 0.05 was considered statistically significant.All statistical analysis was performed with the SPSS version 21  software. The Chi-square test or Fisher’s exact test was used for evaluating the correlation between clinicopathological characteristics and c-MYC and ß-catenin expression. The Pearson correlation coefficient was used for analyzing comparison of detection methods. The Kaplan-Meier method with the log-rank test and multivariate regression were performed to assess survival difference. The survival results were determined with hazard ratio (HR) and its 95 % confidence interval (CI). c-MYC mRNA in situ hybridization score of 0 was observed in 34 (9.3 %), a score of 1 in 92 (25.1 %), a score of 2 in 123 (33.5 %), a score of 3 in 93 (25.3 %), and a score of 4 in 25 (6.8 %). Consequentially, overexpression of c-MYC mRNA (a score of 2–4) was observed in 241 patients (65.7 %). c-MYC protein overexpression was observed in 201 (54.8 %), and ß-catenin nuclear overexpression was observed in 221 (60.2 %) patients.In 367 patients (cohort 1), a P < 0.001, P = 0.007, P = 0.025 and P < 0.001, respectively). In addition, c-MYC protein overexpression was associated with a tumor location in the recto-sigmoid colon. Increased levels of the c-MYC mRNA transcript were associated with microsatellite stable CRC (P = 0.019), located in the sigmoid colon and rectum, and with less aggressive features, similarly to c-MYC protein overexpression. Likewise, ß-catenin nuclear expression was frequently detected in tumors of the recto-sigmoid colon, of low-grade differentiation (P = 0.006), of small size (P = 0.007) and microsatellite stable CRC (P < 0.001).Table P < 0.001), which was classified as moderate correlation [P < 0.001 and 0.211; P < 0.001, respectively).In cohort 1, c-MYC protein overexpression was correlated with mRNA overexpression . All 176 cases had distant metastatic lesions. Among them, 142 cases had lymph node metastases, even though we dissected more than 20 lymph nodes in all CRC patients respectively. The clinicopathological features of the cohort 2 are indicated in Table . All 176In the primary tumors of cohort 2, c-MYC protein overexpression, mRNA overexpression and nuclear ß-catenin expression was detected in 57.6 % (102 out of 176), 77.4 % (137 out of 176) and 61.0 % (108 out of 176) of tumors, respectively. In distant metastatic tumors, c-MYC protein overexpression, mRNA overexpression, and nuclear ß-catenin expression was detected in 37.3 % (66 out of 176), 74.6 % (132 out of 176) and 47.5 % (84 out of 176) of tumors, respectively. In 142 lymph node metastases, we performed c-MYC and ß-catenin analysis in 111 cases which paraffin blocks were available. c-MYC protein overexpression, mRNA overexpression, and nuclear ß-catenin expression was detected in 66.7 % (74 out of 111), 77.5 % (86 out of 111) and 58.6 % (65 out of 111) of tumors, respectively.c-MYC mRNA overexpression between the primary and distant metastatic cancer was detected in 25.6 % (45 out of 176) of cases, and discordance between the primary and lymph node metastatic cancer was observed in 30.6 % (34 out of 111) of cases. Discordance of nuclear ß-catenin expression between the primary and distant metastatic cancer was detected in 29.0 % (51 out of 176) of cases, while discordance between the primary and lymph node metastatic cancer was observed in 26.1 % (29 out of 111) of cases. Consequently, locational heterogeneity of c-MYC and ß-catenin expression was frequently seen in advanced CRC.The locational heterogeneity of c-MYC and ß-catenin status is demonstrated in Table P = 0.011, P = 0.012 and P = 0.033, respectively). When prognostic analysis was performed using the combined status of c-MYC and ß-catenin expression, positivity for both proteins (c-MYC/ß-catenin: +/+) was observed 84/367 (22.9 %) cases and was significantly correlated with an improved survival (P = 0.012). We additionally investigated the c-MYC protein overexpression by density of staining - scoring each tumor as low (0–1) to high (2–3) in cohort 1. The percentage of positive neoplastic cells was correlated with density of staining of neoplastic cells , which was categorized as strong correlation [P = 0.070, Additional file All CRC patients of our study were successfully included survival analysis , but mRNA overexpression was not (P = 0.258). Co-expression of c-MYC and ß-catenin (c-MYC/ß-catenin: +/+) also predicted better prognosis (P = 0.001). The c-MYC and ß-catenin expression status in distant or lymph node metastatic cancer did not significantly associated with patients’ survival . The presence of locational heterogeneity of c-MYC and ß-catenin expression was not associated with survival . In addition, we evaluated the Kaplan–Meier survival for MSI status, stage, chemotherapy status, site of primary cancer and site of distant metastasis , the median follow-up was 43 months (1–105 months), as previous reported . In the , the medP = 0.048 and P = 0.002, respectively). However, individual analysis of c-MYC protein overexpression, mRNA overexpression, and nuclear ß-catenin expression did not independently predict better prognosis.The multivariate Cox’s proportional hazards regression model of c-MYC and ß-catenin expression was described in Table c-MYC gene amplification tend to be correlated with poor survival [c-MYC mRNA overexpression in CRC was reported to be correlated with improved survival [There were several studies on c-MYC status in various malignancies. Some malignant tumors with c-MYC overexpression including gastric carcinoma, esophageal squamous cell carcinoma, and soft tissue leiomyosarcoma are associated with poor survival –34. Likesurvival –37. Intesurvival , but thisurvival . Neverthsurvival . In addisurvival . Recentlsurvival . Howeversurvival , 41.In our study, overexpression of c-MYC protein in the consecutive cohort was significantly correlated with an improved prognosis in univariate model, but not in multivariate model. The prognostic significance of nuclear ß-catenin expression is similar to that of c-MYC protein overexpression. We performed a combined analysis of c-MYC and ß-catenin expression because these proteins are closely related. Astonishingly, co-expression of c-MYC and ß-catenin correlated with an improved prognosis by univariate and multivariate analysis. Although the advanced cohort was mainly consisted of stage IV CRC patients , co-expression of c-MYC and ß-catenin was independently predicted favorable prognosis. Furthermore, overexpression of c-MYC and ß-catenin—except c-MYC mRNA—in the advanced cohort was significantly correlated with better prognosis using a univariate model. Consequently, co-expression of c-MYC and ß-catenin determined by IHC might be of use in the assessment of CRC patients.P < 0.001). Overexpression of c-MYC can be caused by complex regulatory pathways and multiple communications with other factors, rather than just c-MYC gene alterations [c-MYC [APC gene mutation is the initial step of CRC oncogenesis [et al. reported that CpG island methylation interrupts several Wnt target genes, including ASCL2 and LGR5 during CRC progression and promoter methylation of Wnt target genes is a powerful predictive factor for CRC relapse [We also demonstrated that ß-catenin nuclear expression significantly associated with its target molecule c-MYC in CRC patients (ρ, 0.282; erations . An examerations , 43. ß-cs [c-MYC . These pogenesis  and ofteogenesis . Thus, ßogenesis , and indogenesis , 48. For relapse . Therefo relapse , 46–48. In the advanced cohort, regional heterogeneity of c-MYC and ß-catenin expression was frequently observed in advanced CRC. Nonetheless, c-MYC and ß-catenin expression was associated with better prognosis in the primary, not distant and lymph node metastatic cancer. Consequently, when we evaluate prognosis with c-MYC and ß-catenin, tissue from primary CRC should be used. In daily practice for pathologists, metastatic cancer tissue can only occasionally be obtained. Some researchers suggest that regional heterogeneity should be considered as a potential limitation to the evaluation of prognostic and therapeutic value in tissue from metastatic lesions , 50. TheOur study comprehensively evaluated the c-MYC and ß-catenin status of CRC patients. Overexpression of c-MYC protein, mRNA, and ß-catenin nuclear expression were observed in 54.8, 65.7, and 60.2 % of consecutive CRC patients, respectively. c-MYC protein overexpression was significantly correlated with mRNA overexpression and with ß-catenin nuclear expression. Interestingly, co-expression of c-MYC and ß-catenin—in other words, c-MYC overexpression via ß-catenin—was an independent improved prognostic factor in both the consecutive and advanced cohort. These findings indicate that c-MYC and ß-catenin IHC can be used as prognostic marker of CRC patients. However, further investigations on the detailed mechanism of connection between c-MYC and ß-catenin and its impact on patients’ outcome in CRC are needed.


Hyaluronan And Proteoglycan Link Protein 1 (HAPLN1), that appeared de novo in aggressive HCCs expressing cytoplasmic β-catenin and stem cell markers. HAPLN1 was independently associated with bad overall and disease-free outcome. In vitro, HAPLN1 was expressed de novo in EPCAM−/NCAM+ mesoderm-committed progenitors, upon spontaneous epithelial-mesenchymal transition and de-differentiation of hepatocyte-like cells to liver progenitors. In these cells, HAPLN1 knockdown downregulated key markers of mesenchymal cells, such as Snail, LGR5, collagen IV and α-SMA. In conclusion, HAPLN1 reflects a signaling network leading to stemness, mesenchymal commitment and HCC progression.About 20% hepatocellular carcinomas (HCCs) display wild-type β-catenin, enhanced Wnt signaling, hepatocyte dedifferentiation and bad outcome, suggesting a specific impact of Wnt signals on HCC stem/progenitor cells. To study Wnt-specific molecular pathways, cell fates and clinical outcome, we fine-tuned Wnt/β-catenin signaling in liver progenitor cells, using the prototypical Wnt ligand Wnt3a. Cell biology assays and transcriptomic profiling were performed in HepaRG hepatic progenitors exposed to Wnt3a after β-catenin knockdown or Wnt inhibition with FZD8_CRD. Gene expression network, molecular pathology and survival analyses were performed on HCCs and matching non-tumor livers from 70 patients by real-time PCR and tissue micro-array-based immunohistochemistry. Wnt3a reprogrammed liver progenitors to replicating fibrogenic myofibroblast-like cells displaying stem and invasive features. Invasion was inhibited by 30 nM FZD7 and FZD8 CRDs. Translation of these data to human HCCs revealed two tight gene networks associating cell surface Wnt signaling, stem/progenitor markers and mesenchymal commitment. Both networks were linked by  Adenomatous polyposis coli (APC)-axin platform, thus halting GSK3B-dependent phosphorylation of β-catenin at key residues in exon 3. Non-phosphorylated β-catenin is thus stabilized, accumulates in the cytoplasm and nucleus and interacts with T-cell factor (TCF) transcription factors of target gene expression. Conversely, in the absence of interaction of Wnt ligands with their cell surface receptors, phosphorylated β-catenin undergoes proteasomal degradation. β-catenin exon 3 mutations in tumors bypass the GSK3B gatekeeper, thus switching the pathway to the ON position constitutively ,irst 8 h .To look at the gene networks leading to myofibroblastic differentiation of hepatic progenitors, we treated cells with 7 nM Wnt3a for three days. To identify β-catenin-dependent or FZD8_CRD sensitive gene networks, we transfected cells with β-catenin-targeting siRNA or treated them with the prototypical Wnt inhibitor FZD8_CRD. This strategy was validated by real-time PCR gene expression analysis of the β-catenin target gene LGR5 in HepaRG and Huh7 cells . A wholeIngenuity Pathway Analysis revealed fibrogenesis and myofibroblast activation pathways Figure . ConsistTo strengthen our observations, we compared the Wnt-induced signature in HepaRG cells with the whole genome mRNA expression profiles of epithelial 3P and fibroblast-like 3SP human HCC cell lines. These cells are derived from the same HCC, 3SP resulting from 3P cells that underwent spontaneous EMT . Gene SeClose analysis of Wnt-induced genes sensitive to both β-catenin targeting siRNA and to FZD8_CRD  revealedHyaluronan And Proteoglycan Link Protein 1 (HAPLN1) is a cartilage gene that links proteoglycans to hyaluronic acid, thereby playing a key role in extracellular matrix assembly [ were binarized by a ≥3 cut-off score for focal expression of specific proteins.After removing experimental batch effect with the  package , gene ne package  using ex package . To anal package . Briefly package , the incsurvival, FactoMineR, gplots, colorspace and marray or with Statistica 10 .Correlation analyses between gene expression levels were performed with the Spearman rank order test . Association analyses between binary or ordinal variables  were performed with Pearson's Chi2 or Fisher exact test. Group comparisons were done with non-parametric ANOVA and the Kruskal Wallis post hoc tests, unless otherwise indicated. Heatmaps showing associations between binary patient data were constructed with Excel (Microsoft Office 2010). Statistical calculations were performed with the R software (version 3.1.1) and the packages See


Non-verbal social interaction between humans requires accurate understanding of the others’ actions. The cognitivist approach suggests that successful interaction depends on the creation of a shared representation of the task, where the pairing of perceptive and motor systems of partners allows inclusion of the other’s goal into the overarching representation. Activity of the Mirror Neurons System (MNS) is thought to be a crucial mechanism linking two individuals during a joint action through action observation. The construction of a shared representation of an interaction  depends upon sensorimotor cognitive processes that modulate the ability to adapt in time and space. We attempted to detect individuals’ behavioral/kinematic change resulting in a global amelioration of performance for both subjects when a common representation of the action is built using a repetitive joint action. We asked pairs of subjects to carry out a simple task where one puts a base in the middle of a table and the other places a parallelepiped fitting into the base, the crucial manipulation being that participants switched roles during the experiment. We aimed to show that a full comprehension of a joint action is not an automatic process. We found that, before switching the interactional role, the participant initially placing the base orientated it in a way that led to an uncomfortable action for participants placing the parallelepiped. However, after switching roles, the action’s kinematics by the participant who places the base changed in order to facilitate the action of the other. More precisely, our data shows significant modulation of the base angle in order to ease the completion of the joint action, highlighting the fact that a shared knowledge of the complete action facilitates the generation of a common representation. This evidence suggests the ability to establish an efficient shared representation of a joint action benefits from physically taking our partner’s perspective because simply observing the actions of others may not be enough. However, social communication is not limited to just verbal communication. Through their behavior, humans change their environment, satisfy internal needs, and achieve personal goals . Since mirror neurons (MN) were discovered more than 20 years ago, we have been able to apply a neuroscientific approach and a new understanding of social interaction. Initially discovered in non-human primates  and in hIn recent years, great progress has been made by investigating neural processes during interpersonal motor interactions . These sIn line with this model, it is our opinion that building experimental paradigms allowing bidirectional and either synchronic or diachronic adaption  involvinphysically experienced the other partner’s motor task difficulty. Results indicate that the adaptation to the new task was not automatic and required a common experience from both participants. We wish to point out a limit of the maximalist interpretations of the MNS, according to which this system serves the understanding of others action and provide empirical data supporting the minimalist approach of the representational content of MN  when both individuals had nt of MN . AlthougTwenty healthy right-handed subjects  participated in the Main experiment and 10 healthy right-handed subjects participated in the Control Experiment . None of them had any history of neurological disorder. All participants gave written consent before the experiment. Participants were familiarized with the methods prior to the experiment. and an explanation of the purpose of the study was given at the end of the experiment. The study was approved by the Ethical Committee CPP Sud-Est II.Figure 1). The subjects were asked to carry out a simple task: S1 was to move the base to the center of the table and S2 was to place the parallelepiped in the slot on the base. Note that the action performed by S2 is more or less facilitated depending on how S1 place the base on the table: difficult when the slot in the base remains parallel to the sagittal axis; easy when it is slightly tilted to the left. The experiment was divided into three conditions with instructions given prior to each condition. In Condition 1, the joint action was repeated 20 times with no other instruction other than completing the task after a vocal “Go” signal from the experimenter, which was the same for every trial. In Condition 2, subject’s roles were interchanged so S1 was in charge of moving the parallelepiped and S2 the base for 3 trials. Because this second condition was designed to give the opportunity for participants to experience the other’s action, data from Condition 2 was not analyzed. In Condition 3, subjects reverted to their initial roles (so they were carrying out the same task as in Condition 1) for another 20 trials. In this final condition, participants were further instructed to perform the task as fast as possible. During the whole experiment, participants were not allowed to communicate their feelings either verbally or by explicit non-verbal utterance. To ensure the accuracy and well-being of the experiment, an experimenter continuously stayed next to the table in order to both control inter-subjects communication and to replace objects at their correct initial location when required. At no time were participants encouraged to change their motor behavior either explicitly or implicitly; they believed the goal was a simple kinematic study until the end of the experiment.The experiment involved a pair of participants: subject 1 (S1) and subject 2 (S2). In the following, participants will be referred as S1 or S2 depending on their original role in the experiment (Condition 1). Subjects were seated face to face on opposite sides of a 50 cm × 70 cm table. Both subjects were instructed to use only their thumbs and index fingers to grip and displace the experimental objects, which were placed on predefined spots: the cylindrical base located to the right of S1 and the parallelepiped to the right of S2. The slot of the base and the parallelepiped were both oriented parallel to the sagittal axis . This control served to ascertain that verbal instruction to perform the task as fast as possible was not sufficient to provoke potential behavioral changes. A critical point of the Control experiment was that participants were only assigned to one role; they did not have the opportunity to experience the other participant’s task.The movements of the right arm and hand of both subjects were recorded by means of an Optotrak Certus camera . The camera was fixed 2 m away from the table. Five markers were placed on each subject: marker 1 was on the distal extremity of the thumb, marker 2 on the distal extremity of the index, marker 3 on the metacarpophalangeal joint of the index, marker 4 on the radial styloid and marker 5 was 3 cm over the radial styloid. The spatial position of active markers was sampled at 250 Hz with a spatial precision of 0.1 mm.Reach-to-Grasp and Displace). Sub-movements onset and offset were determined by a sequence of at least eleven increasing or decreasing points of the wrist marker velocity profiles. Velocity peak was determined as the maximal value in wrist marker velocity profiles. The workspace was defined by the X, Y, Z axes defined by the table surface. The angle between the fingers markers 1 and 2 of S2 and the sagittal axis was also analyzed. This angle corresponded to the opposition axis . Kinematic parameters were assessed for each individual movement using Optodisp software .Kinematic parameters were determined for each individual trial and averaged for each participant and condition. Statistical analysis data was analyzed by using Statsoft Statistica 8. General Linear Model (GLM) and the Greenhouse–Geisser correction for non-sphericity was applied when appropriate . Post hoExecution time and Velocity peaks were both analyzed each by separated 2×2×2 (Condition × Movement × Subject) ANOVAs and final angles by a one-way ANOVA.F = 70,824, p < 0,001], indicating a smaller execution time during Condition 3 compared to Condition 1. There was a significant main effect of movement , showing that Reach-to-Grasp movement was realized faster than Displace and a significant main effect of subject  that showed that S1’s action were executed faster than S2’s. Post hoc test indicated that S1 execution times were smaller in Condition 3 compared to Condition 1 for both Reach-to-Grasp  and Displace   and that S2 execution times were smaller in Condition 3 compared to Condition 1 for both Reach-to-Grasp  and Displace  .The 2 × 2 × 2 (Condition × Movement × Subject) ANOVA showed a significant main effect of Condition , indicating a greater execution speed during Condition 3 compared to Condition 1. A significant main effect of movement  showed that Reach-to-Grasp movement was realized with a smaller speed than Displace. Post hoc test indicated that S1 velocity peaks were greater in Condition 3 compared to Condition 1 for both Reach-to-Grasp  and Displace   and S2 velocity peaks were greater in Condition 3 compared to Condition 1 for both Reach-to-Grasp  and Displace  .The 2 × 2 × 2 (Condition × Movement × Subject) ANOVA showed a significant main effect of Condition . Post hoc test revealed that the final angle in Condition 3 was significantly smaller compared to Condition 1  and compared to Control  .In order to compare the final angles of Condition 1, Condition 2, and Control, we performed a one way ANOVA that showed a main effect [Reach-to-Grasp and Displace, for both participants when common representation of the task was achieved.In this paper, we developed a diachronic joint action task that induced behavioral adjustments made by one of the participants in order to make the task of their partner easier and consequently improving the common realization of the task for both participants. The results revealed better performance (execution time and velocity) in both sub-movements of the task, Displace sub-movement: the articular tension in the arm and wrist joints are less extreme in Condition 3 than in Condition 1. This behavioral measurement indicates that the participant placing the base changed their previous behavior, after experiencing the partner’s contribution to the joint action.Our measurements also highlight a significant change in the final angle of the cylindrical base between the Condition 1 (before the subjects switched roles) and Condition 3 (after the subjects switched their roles). According to d) according to d values calculated for velocity peaks for each sub-movement in Condition 1 and Condition 3 for S2 . Cohen’s d value for Reach-to-Grasp sub-movement between Condition 1 and Condition 3 is 0.76, corresponding to a medium effect, while Cohen’s d for Displace Sub-movement between Condition 1 and Condition 3 is 1.38, corresponding to a large effect  changed the orientation of the base. On the one hand, imposed constrain on movement speed in Condition 3 improves the kinematics parameters in both sub-movements but on the other hand, we observe a greater orientation-specific effect only on the Displace sub-movement. This result is consistent with previous data from The results from our statistical analysis reveal an increase in the performance of the subjects for both sub-movements during Condition 3. In Condition 3, both subjects were asked to perform the action as fast as possible. Due to this, one can assess that the instruction (to perform the task as fast as possible) is the main contributor to the increase in performance, rather than changes in the orientation. Because of this, we were also able to calculate the effect size . This change in the instructions was only to maintain participants focus and avoid boredom.From the neural perceptive, parietal and premotor regions are thought to form the action observation network, also known as the MNS . AlthougOver the last few decades, scientific research has improved our understanding of how perception and action are linked. It still remains unclear whether the processing of relevant information during joint actions emerges from the physical and informational constraints  or whethjoint Simon effect arises when performed by a pair of participants (where each participant has a go/no-go task). The joint Simon task has been used to highlight co-representations during joint tasks, suggesting the existence of a specific neural mechanism facilitating social interactions with conspecifics (One of the most studied joint tasks is derived from the Simon task  where papecifics . An altepecifics  and furtpecifics . The finpecifics .All authors provided substantial contributions to the conception or design of the work. QM and LG were in charge of the acquisition of data, QM, LG, and YP were in charge of the analysis, all authors were involved in the interpretation of data for the work. QM and YP drafted the work and TN revised it critically and all authors gave final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


AKR1B1 to human-relevant levels, mice devoid of Akr1b3, and pharmacological inhibitors of AR, we demonstrated that AR is an important component of myocardial I/R injury and that inhibition of this enzyme protects the heart from I/R injury. In this study, our objective was to investigate if AR modulates the β-catenin pathway and consequent activation of mesenchymal markers during I/R in the heart. To test this premise, we used two different experimental models: in vivo, Akr1b3 null mice and wild type C57BL/6 mice (WT) were exposed to acute occlusion of the left anterior descending coronary artery (LAD) followed by recovery for 48 hours or 28 days, and ex-vivo, WT and Akr1b3 null murine hearts were perfused using the Langendorff technique (LT) and subjected to 30 min of global (zero-flow) ischemia followed by 60 min of reperfusion. Our in vivo results reveal reduced infarct size and improved functional recovery at 48 hours in mice devoid of Akr1b3 compared to WT mice. We demonstrate that the cardioprotection observed in Akr1b3 null mice was linked to acute activation of the β-catenin pathway and consequent activation of mesenchymal markers and genes linked to fibrotic remodeling. The increased activity of the β-catenin pathway at 48 hours of recovery post-LAD was not observed at 28 days post-infarction, thus indicating that the observed increase in β-catenin activity was transient in the mice hearts devoid of Akr1b3. In ex vivo studies, inhibition of β-catenin blocked the cardioprotection observed in Akr1b3 null mice hearts. Taken together, these data indicate that AR suppresses acute activation of β-catenin and, thereby, blocks consequent induction of mesenchymal markers during early reperfusion after myocardial ischemia. Inhibition of AR might provide a therapeutic opportunity to optimize cardiac remodeling after I/R injury.Aldose reductase , the first enzyme in the polyol pathway, plays a key role in mediating myocardial ischemia/reperfusion (I/R) injury. In earlier studies, using transgenic mice broadly expressing human  Acute myocardial infarction (AMI) remains the leading cause of morbidity and mortality worldwide . The extin vivo model of I/R, and an ex vivo intact heart preparation subjected to I/R. We employed mice devoid of AR (Akr1b3 null mice) to determine whether altered flux via AR influences myocardial β-catenin during I/R. Our results indicate that genetic deletion of Akr1b3 drives acute induction of mesenchymal markers, at least in part via induction of the β-catenin pathway during I/R in the heart.The Wnt/β-catenin pathway plays an important role in various biological processes including development, differentiation, proliferation and tissue homeostasis , 19. ActAkr1b3 null mice to left anterior descending coronary artery (LAD) occlusion for 30 min, followed by 48 hours of reperfusion (LADp48h). The expression or absence of AKR1B3 in WT and Akr1b3 null mice, respectively, was verified by Western blot  –33. Sevee hearts  leads toPecam1, cadherin 5 (Cdh5) and claudin 5 (Cldn5), with concomitant activation of mesenchymal markers (α-smooth muscle actin (Smaa), Transgelin (Sm22), Vimentin (Vim), S100a4 (Fsp1) and fibronectin 1 (Fn1) and the related EndMT transcription factors (Snai1 and Snai2). Analysis of the transcription factors involved in EndMT, Snai1 and Snai2 revealed upregulation of Snai1, but not Snai2 in hearts devoid of Akr1b3 , which were transfected with siRNA against Akr1b3 (Abr1b3 siR). Scrambled siRNA was used as a negative control (scr siR). 90% knockdown efficiency was achieved in these cells  suggested an activated fibroblast phenotype in endothelial cells derived from mice devoid of Akr1b3.EndMT is characterized by reduction of endothelial markers platelet/endothelial adhesion molecule  and MMPs (Mmp2 and Mmp9) revealed an increase in both types of collagens as well as MMPs in mice hearts devoid of Akr1b3 after I/R .Post-infarction remodeling is a complex process in which cardiomyocyte loss is accompanied by cellular hypertrophy and fibrosis. Initially an adaptive response, extended cardiac fibrosis can lead to maladaptive remodeling and heart failure. Our results indicate that there was no evidence of hypertrophy in either WT or f Akr1b3 . Furtherpost-LAD . Furtherpost-LAD . We measn levels . Taken tAkr1b3 was due to enhanced β-catenin levels, we employed IWR-endo, an inhibitor of Wnt-β catenin pathway [ex-vivo isolated perfused heart for model of I/R, Akr1b3 null mice hearts were treated with either vehicle or IWR-endo during the 30 min of ischemia and 60 min of reperfusion. qRT-PCR analysis from the RNA extracted from heart tissues revealed that IWR-endo treatment reduced the expression of mesenchymal and fibrotic markers after I/R in these mice hearts when compared to the vehicle treatment  and fibroblast activation. Akr1b3 null mice hearts displayed enhanced expression of mesenchymal markers, suggestive of fibroblast activation. Several studies had suggested the role of β-catenin in fibrosis and mesenchymal activation as a key driver of wound healing [Akr1b3 null hearts and that these increases require β-catenin. healing –44. Publ healing , 46. ConAkr1b3 during I/R in our study could be attributed to either TGFB2 or GSK3B phosphorylation or both. Studies have shown that activation of β-catenin could be due, in part, to phosphorylation of GSK3β. The phosphorylation of GSK3β by p-Akt destabilizes GSK3β complex, resulting in the dissociation of β-catenin from the GSK3β complex. This process leads to nuclear translocation of β-catenin [Akr1b3-/- hearts compared to WT hearts (14), here we focused on the role of TGFB2. Since TGFB2 has been implicated in β-catenin activation and cellular plasticity [Akr1b3 could also be due to increases in TGFB2. Our studies in cardiac fibroblasts from WT and Akr1b3 null mice subjected to H/R revealed only modest activation of mesenchymal markers in the presence of TGFB2, indicating other mechanisms are also contributing to mesenchymal activation. Specifically, in addition to TGFB2, secreted factors from other cells in heart may play an important role in activation of mesenchymal markers in our study.Activation of β-catenin in the mice hearts devoid of -catenin , 47. Ear-catenin , 27, 48.asticity , 30, it Akr1b3-/- hearts. Repair mechanisms to manage tissue scaring are essential for viable recovery after I/R. We believe β-catenin related changes may be of help in reducing the tissue scar through mesenchymal activation. In our studies we show that CF activation results in upregulation of both matrix metalloproteinases and collagens in Akr1b3-/- hearts, key players that facilitate removal and replacement of necrotic tissue. Hence we posit, in Akr1b3-/- mice, TGFB2 mediated mesenchymal activation may aid in the clearance of necrotic tissue thereby reducing injury.The impact of mesenchymal activation on cardiac remodeling post infarction may, in part, explain reduced I/R injury in Mmp knockout  mice showed cardioprotection, inhibitor studies as well as macrophage-specific MMP9 overexpression studies contradicted the earlier observations [Mmp2, Mmp9, as well as collagenases, without increases in the total collagen content in the mice hearts devoid of Akr1b3 in I/R. Hence our findings on MMP changes are those in line with macrophage specific MMP9 overexpression mice and MMP9 inhibitor studies [MMPs are involved in physiological as well as pathological processes, such as inflammation, tumor metastasis and tissue remodeling . Severalrvations , 55. Suc studies , 55.Akr1b3 null mice in acute (48 hours) as well as extended periods of recovery (28 days) was linked to β-catenin changes. Our study identifies that β-catenin-mediated mesenchymal activation is linked to reduced myocardial I/R injury and that this pathway is downstream of the AKR1B3 pathway. In myocardial I/R, deletion of Akr1b3 modulates the myocardial β-catenin pathway and consequent induction of mesenchymal markers as well as genes linked to fibrotic remodeling, thereby highlighting inhibition of the AKR1B3 pathway as a key strategy in protection against myocardial injury after infarction.Several studies point out the importance of prolonged structural alteration resulting in fibrosis and heart failure. TGFB2 and β-catenin mediated changes in our studies are likely related to acute structural alterations. Studies in the literature are mixed in regard to the role of TGFB in I/R hearts, with some studies showing the detrimental effects of TGFB and others showing cardioprotective effects of TGFB during I/R. –60 LikewAkr1b3 null mice- and wild type littermate mice were used as described earlier [All animal experimentations were performed with the approval of the Institutional Animal Care and Use Committee at New York University School of Medicine. Male  earlier , 61. Sur earlier . Briefly earlier , 63.2>600 mm Hg was maintained in the oxygenation buffer. IWR-endo was added at 10μM final concentration to the buffer, whereas DMSO was added for vehicle control. Hearts were perfused with IWR or vehicle starting at 10 min prior to initiation of ischemia for 30 min and continued throughout 60 min of reperfusion.Experiments were carried out as described earlier , 15. Briin vivo) and effluents (ex vivo) were measured using a commercially available kit .Lactate dehydrogenase (LDH) levels in plasma  for 30 min of hypoxia  at 37°C followed by 60 min of reoxygenation in 5% CO2 incubator . Cells were collected at the end of reoxygenation in ice cold PBS for further analysis.For transfection in mouse aortic endothelial cells, siRNA specific against Murine primary cardiac fibroblasts were isolated using the protocol mentioned . BrieflyTotal collagen levels from heart tissues subjected to LAD post 48hrs and 28 days were measured using total collagen assay kit (Biovison). Briefly, tissues were homogenized in concentrated HCl and hydrolyzed at 120°C for 3 h. Hydrolyzed sample were vacuum evaporated and subjected to quantification as per manufacturer‘s protocol.Total lysates from heart tissues or cells were prepared using lysis buffer . Tissues/cells were crushed using beads for homogenization and were quantified. For the detection of the protein following antibodies were used β-catenin, RUNX2 , TGFB2 (Santacruz), MMP2 (abcam), β-ACTIN (sigma) and ARKR1B3 (GeneTex). Antibodies were used at a final concentration of 1μg/ml.cDNA was prepared from whole heart tissues or cells using RNAeasy isolation kit (Qiagen). cDNA was prepared using iScript cDNA synthesis kit (BioRad). Fast sybrgreen mastermix and Taqman probes were used for quantification and data were normalized using 18s rRNA. Details of the primers used are in All values are presented as the mean ± standard error of the mean. Data were analyzed by unpaired two-tailed t-tests to assess the difference between groups as specified. A probability value of ≤ 0.05 was considered significant.S1 Table(PDF)Click here for additional data file.


Gliomas cells are the site of numerous metabolic and thermodynamics abnormalities with an increasing entropy rate which is characteristic of irreversible processes driven by changes in Gibbs energy, heat production, intracellular acidity, membrane potential gradient, and ionic conductance. We focus our review on the opposing interactions observed in glioma between the canonical WNT/beta-catenin pathway and PPAR gamma and their metabolic and thermodynamic implications. In gliomas, WNT/beta-catenin pathway is upregulated while PPAR gamma is downregulated. Upregulation of WNT/beta-catenin signaling induces changes in key metabolic enzyme that modify their thermodynamics behavior. This leads to activation pyruvate dehydrogenase kinase 1(PDK-1) and monocarboxylate lactate transporter 1 (MCT-1). Consequently, phosphorylation of PDK-1 inhibits pyruvate dehydrogenase complex (PDH). Thus, a large part of pyruvate cannot be converted into acetyl-CoA in mitochondria and in TCA (tricarboxylic acid) cycle. This leads to aerobic glycolysis despite the availability of oxygen, named Warburg effect. Cytoplasmic pyruvate is, in major part, converted into lactate. The WNT/beta-catenin pathway induces also the transcription of genes involved in cell proliferation, cell invasiveness, nucleotide synthesis, tumor growth, and angiogenesis, such as c-Myc, cyclin D1, PDK. In addition, in gliomas cells, PPAR gamma is downregulated, leading to a decrease in insulin sensitivity and an increase in neuroinflammation. Moreover, PPAR gamma contributes to regulate some key circadian genes. Abnormalities in the regulation of circadian rhythms and dysregulation in circadian clock genes are observed in gliomas. Circadian rhythms are dissipative structures, which play a key role in far-from-equilibrium thermodynamics through their interactions with WNT/beta-catenin pathway and PPAR gamma. In gliomas, metabolism, thermodynamics, and circadian rhythms are tightly interrelated. Gliomas are the most frequent primary brain tumors. Around 30 percent of all brain and central nervous system tumors and 80 percent of all malignant brain tumors are gliomas. They are characterized by their infiltrating nature, especially into the surrounding normal brain tissue , while the activation of PPAR gamma induces inhibition of canonical WNT/beta-catenin pathway  are dissipative structures, which play a key role in far-from-equilibrium thermodynamics. In gliomas, abnormalities in the regulation of CRs are observed  and disruption of circadian rhythms. The thermodynamic dysregulation induced by these two processes is consubstantial with metabolic abnormalities found in glioma.We focus this review on the opposing interactions observed in glioma between the canonical WNT/beta-catenin pathway and PPAR gamma and their metabolic and thermodynamic implications.The canonical WNT/beta-catenin pathway plays an important role in metabolism, embryonic development, cell fate, and epithelial-mesenchymal transition (EMT). The canonical WNT activity is reflected by elevated levels of beta-catenin in the nucleus and/or cytoplasm, which can be detected by means of immunohistochemical staining and Western blotting. Its dysfunction is involved in numerous diseases, particularly in cancers . PPAR gamma is expressed in numerous cell types, such as adipose tissues, muscles, brain, and immune cells. PPAR gamma activates the expression of many genes and regulates glucose homeostasis, insulin sensitivity, lipid metabolism, immune responses, cell fate, and inflammation , since the pathway via free fatty acid beta-oxidation is less efficient (ATP/O2 = 5.6). This takes about 11% more O2 to produce the same amount of ATP from fatty acids as it does from glucose. Moreover, PDK-1 and 2 enhance angiogenesis , an allosteric enzyme, is responsible for glycolytic oscillations. PFK can lead to instabilities beyond which a new state can be organized in time and in space  is a type of receptor tyrosine kinase (RTK) playing a central role in cell division, migration, adhesion, differentiation, and apoptosis  is converted in PIP3  by PI3K and back by phosphatase and tensin homolog (PTEN)  by inducing HIF-1alpha, which suppresses glucose entry into the TCA cycle  by activating the WNT/beta-catenin pathway. The link between PGE2 and the canonical WNT pathway suggests that chronic inflammation induced by a prolonged increase of PGE2 could lead to activation of WNT signaling resulting in cell proliferation and cancer. PGE2 enhances the beta-catenin-dependent transcription  can reduce nuclear beta-catenin levels and induce beta-catenin degradation  in zebrafish. Inhibition of PGE2 synthesis blocks alterations in HSC induced by WNT. PGE2 modifies the WNT signaling cascade at the level of beta-catenin degradation through the cAMP/PKA pathway. WNT activation in stem cells requires PGE2 , is known to drive numerous biologic processes in the body. CRs can be defined as endogenous, entrainable free-running periods that last ~24 h. CRS are far-from-equilibrium dissipative structures and are due to a negative feedback produced by a protein on the expression of its own gene Goodwin, . They opCRs govern numerous physiological and metabolic functions  (Soták et al., Beta-catenin induces Per2 degradation altering circadian clock gene in intestinal mucosa of ApcMin/+ mice (Yang et al., PPAR interferes with the mammalian clock and energy metabolism (Chen and Yang, Gliomas exhibit thermodynamic and metabolic alterations and abnormal circadian rhythms with an increasing entropy rate. In gliomas, the canonical WNT/beta-catenin pathway is upregulated, while PPAR gamma is downregulated. The two systems act in an opposite manner. Overactivation of the WNT pathway results in cell proliferation, due to the activation of target genes of beta-catenin, such as cyclin D1 and c-Myc. This activation of WNT pathway also promotes protein synthesis and angiogenesis. PDK and MCT-1 are also target genes of beta-catenin, explaining the decrease in the transformation of pyruvate into acetyl-CoA in mitochondria and the formation of intracellular lactate, which will be extruded out the cell. This is referred to as aerobic glycolysis or the Warburg effect. Circadian rhythms, dissipative structures, which are governed by the laws for far-from-equilibrium thermodynamics are disrupted in gliomas. They are influenced by both the WNT/beta-catenin pathway and PPAR gamma. Changes in thermodynamics, metabolism, and circadian rhythms are tightly linked in gliomas.All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest. The reviewer DJRL and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


The administration of cisplatin is limited due to its nephrotoxic side effects, and prevention of this nephrotoxicity of cisplatin is difficult. Mesenchymal stem cell (MSC)-derived exosomes have been implicated as a novel therapeutic approach for tissue injury. In this study, we demonstrated that the pretreatment of human umbilical cord MSC-derived exosomes (hucMSC-Ex) can prevent the development of cisplatin-induced renal toxicity by activation of autophagy in vitro and in vivo., we constructed cisplatin-induced acute kidney injury rat models. Prior to treatment with cisplatin for 0.5 h, hucMSC-Ex or HFL1-Ex were injected into the kidneys via the renal capsule. 3-methyladenine and rapamycin were injected under the kidney capsule before hucMSC-Ex. All animals were sacrificed at 3 days after cisplatin injection. Renal function, Luminex assay, tubular apoptosis and proliferation, and autophagy response were evaluated.In vitro, rat renal tubular epithelial (NRK-52E) cells were pre-incubated with exosomes from hucMSC or HFL1  for 30 min, and 3-methyladenine (an autophagic inhibitor) and rapamycin (an autophagic inducer) for 1 h before cisplatin treatment for 8 h, respectively. Cells were harvested for apoptosis assay, enzyme-linked immunosorbent assay (ELISA), Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR). In vivohucMSC-Ex inhibited cisplatin-induced mitochondrial apoptosis and secretion of inflammatory cytokines in renal tubular epithelial cells in vitro. hucMSC-Ex increased the expression of the autophagic marker protein LC3B and the autophagy-related genes ATG5 and ATG7 in NRK-52E cells. Rapamycin mimicked the effects of hucMSC-Ex in protecting against cisplatin-induced renal injury, while the effects were abrogated by the autophagy inhibitor 3-methyladenine in the animals.Our findings indicate that the activation of autophagy induced by hucMSC-Ex can effectively relieve the nephrotoxicity of cisplatin. Therefore, pre-treatment of hucMSC-Ex may be a new method to improve the therapeutic effect of cisplatin.The online version of this article (doi:10.1186/s13287-016-0463-4) contains supplementary material, which is available to authorized users. Administration of a high dose of cisplatin is limited due to its nephrotoxic side effects , 2. Ther healing –17. All Studies have demonstrated that autophagy is critical for normal proximal tubule function and protection against acute tubular injury , 19. TheHerein, we show that hucMSC-Ex had a markedly preventative effect against cisplatin-induced renal toxicity, and we explored the mechanism of action. Our findings indicate that hucMSC-Ex promoted autophagy in renal tubule epithelial cells and kidney tissues by cisplatin induced through the inhibition of mTOR, thus alleviating cell apoptosis and inflammatory response in the early stages.The study was approved by the ethical committee of Jiangsu University (2012258).2. When well-developed colonies of fibroblast-like cells reached 80% confluency, cultures were trypsinized into new flasks for further expansion. hucMSCs were identified by fluorescence-activated cell sorting (FACS) and differentiation experiments.After obtaining parental and ethics committee consent, the fresh umbilical cords were collected and processed within 6 h as described previously . UmbilichucMSCs were resuspended in phosphate-buffered saline (PBS). Cell aliquots (300 μl) were incubated with fluorescein isothiocyanate (FITC)-conjugated or phycoerythrin (PE)-conjugated monoclonal antibodies specific for CD13, CD29, CD44, CD90, CD105, HLA-1, CD45, HLA-DR, and CD34 on ice for 30 min. FITC or PE mouse nonimmune isotypic IgG were used as controls. All antibodies were purchased from BD Company. The pluripotency of hucMSCs was confirmed by their ability to differentiate into osteocytes and adipocytes, as described previously .As control cells, human fetal lung fibroblast-1 (HFL1) cells were obtained from Cell Bank, Type Culture Collection Committee, the Chinese Academy of Sciences . In order to allow exosome separation, HFL1 cells were cultured with serum-free α-modified minimum essential medium (α-MEM); hucMSCs were cultured with serum-free LG-DMEM. The conditioned medium was collected at 48 h. Density gradient centrifugations were executed and the procedure for the extraction and purification of exosomes was performed as described previously , 14. TheIn order to analyze the characteristics of the exosomes, particle size, substantial shape, and the relative-intensity three-dimensional plot of hucMSC-Ex were tested using the NTA system. The purified hucMSC-Ex were applied to glow-discharged carbon-coated copper grids to detect the precise shape. PTA (phosphor-tungsten acid) staining was conducted for 30 min. The grids were then rinsed with droplets of deionized water and dried. Ultra-thin sections of NRK-52E cells were prepared to observe the formation of autophagic bodies. Transmission electron micrographs were recorded using a Tecnai 12 .Normal rat kidney epithelial (NRK-52E) cells were obtained from Cell Bank . NRK-52E cells were pre-incubated with exosomes from hucMSC (200 μg/ml) or exosomes from HFL1 (200 μg/ml) for 30 min, followed by treatment with cisplatin (8 μM) for 8 h. 3-methyladenine  or rapamycin  was given for 0.5 h before exosomes treatment and 1 h before cisplatin treatment in NRK-52E cells, respectively.Female Sprague-Dawley rats at 6–8 weeks old, weighing 210–250 g, were used. The animals were kept under standard laboratory conditions . Animals were divided into seven groups of six rats each and treated as follows: 1) control group (no cisplatin treatment); 2) PBS group ; 3) hucMSC-Ex group ; 4) HFL1-Ex group ; 5) hucMSC-Ex + 3MA group (500 μg/kidney 3MA was injected under the kidney capsule before hucMSC-Ex and cisplatin administration); 6) 3MA group (500 μg/kidney 3MA was injected under the kidney capsule before cisplatin administration); and 7) Rapa group (20 μg/kidney 3MA was injected under the kidney capsule before cisplatin administration).All animals were sacrificed at 3 days after cisplatin injection. Kidney tissue specimens were divided into two. One was quickly excised, rinsed in ice-cold saline, and fixed in 4% paraformaldehyde, and the paraffin-embedded tissues were sliced and stained with hematoxylin and eosin (H&E). Sections were analyzed and at least 20 random fields were scored by a nephropathologist in a blinded manner. Tubular cell necrosis, tubular dilation, and tubular protein casts (200× magnification) in sections were observed and analyzed. Abnormalities were graded by a semiquantitative score from 0 to 4: 0, no abnormalities; 1, changes affecting less than 25% of the sample; 2, changes affecting 25 to 50%; 3, changes affecting 50 to 75%; 4, changes affecting more than 75%. The other specimen was used immediately or frozen at –86 °C until further biochemical analysis. Blood samples were collected before and after cisplatin injection . Serum creatinine (Cr) and blood urea nitrogen (BUN) levels were measured by an automatic biochemical analyzer . Serum levels of inflammatory cytokines such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL6 were determined by Luminex assays .NRK-52E cells seeded in 24-well plates were treated as described for the in vitro experiment. After treatment with cisplatin for 8 h, the cells were fixed with 4% paraformaldehyde. Mitochondrial membrane potential was measured by a JC-1 kit according to the manufacturer’s protocol .Total RNA was extracted with Trizol reagent  from NRK-52E cells and kidney tissues; cDNA was synthesized using a reverse transcription kit according to the manufacturer’s instructions . Quantitative real-time polymerase chain reaction (qRT-PCR) was used to detect the expression of ATG5, ATG7, and β-actin genes. All samples were examined in triplicate, and all reactions were repeated three times independently using the CFX96 Touch™ Real-Time PCR Detection System .Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining was used to detect the apoptotic cells. hucMSCs, HFL1 cells, NRK-52E cells, and the kidney tissues were examined using the TdT-FragEL DNA fragmentation detection kit according to the manufacturer's protocol . Positive cells were identified as dark brown nuclei under a light microscope . The number of apoptotic cells was counted in 20 randomly selected visual fields of blinded samples, at 200× magnification.Cells, exosomes, and tissues were lysed in RIPA buffer. Protein concentration was determined using the BCA assay kit . Sources and dilution factors of primary antibodies were: rabbit polyclonal CD63 , CD9 , CD81 , PCNA , BCL-XL , BCL-2 , Bax , Cytochrome C , caspase-3 , IL-1β , LC3B , Beclin-1 , mTOR , p-mTOR , 4EBP1 , p70S6K , and mouse monoclonal GAPDH . The nucleoprotein and plasma protein was separated by the nuclear and plasma protein isolation kit , with the primary antibody NF-kB-P65 in the nucleus , primary antibody nucleoprotein Histone . After incubation with the primary antibodies overnight at 4 °C, membranes were washed three times with Tris-buffered saline with 0.05% Tween-20 and challenged with HRP-conjugated goat anti-rabbit or goat anti-mouse antibody . Western blot was performed by Luminata™ crescendo western HRP substrate  and analyzed using MD Image Quant Software.NRK-52E cells, treated as described for the in vitro experiment, were fixed in 4% paraformaldehyde and permeabilized with HEPES-Triton X100 buffer. The paraffin-embedded kidney tissue sections were dewaxed. Endogenous peroxidase activity was then inhibited by exposure to 3% hydrogen peroxide for 10 min. Subsequently, the sections were boiled for 10 min in citrate buffer  for antigen retrieval. The sections were then blocked with 5% BSA  and incubated with TNF-α  and PCNA  primary antibody at 37 °C for 1 h. After the sections were washed with PBS, they were then incubated with diluted secondary antibody for 20 min. Finally, cells were visualized using diaminobenzidine (DAB) substrate and counterstained with hematoxylin for microscopic examination. The number of PCNA-positive cells were counted in 20 randomly selected visual fields of blinded samples, at 200× magnification.NRK-52E cells were treated as mentioned above. Culture supernatants were collected and cell debris was removed by centrifugation. Supernatants were examined using the rat TNF-α enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer's protocol .Female Sprague-Dawley rats were treated as mentioned above. The serum inflammatory cytokines TNF-α, IL-1β, and IL6 were measured using Luminex kits  according to the manufacturer’s instructions.NRK-52E cells were seeded in 24-well plates and cultured for 24 h, then transfected with mRFP-GFP-LC3 adenovirus according to the manufacturer's protocol . Then, cells were cultured on coverslips and divided into four groups: control; PBS; pre-treated hucMSC-Ex; and pre-treated HFL1-Ex. After treatment, the cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with HEPES-Triton X100 buffer. Finally, the cells were stained with Hoechst33342 for nuclear staining, and the images were acquired with a structured illumination microscopy .t test or by analysis of variance (ANOVA) with Newmann-Keuls multicomparison or Dunnett’s post hoc tests as appropriate. A two-tailed P value <0.05 was considered statistically significant.All data from different experiments are expressed as mean ± SD. Statistical analysis was performed by Student’s MSCs isolated from the umbilical cord were characterized by FACS analysis  as follows: 1) control group ; 2) PBS group ; 3) hucMSC-Ex group (hucMSC-Ex (200 μg/kidney) were injected under bilateral renal capsules before treatment with cisplatin); and 4) HFL1-Ex group  was injected under the kidney capsule before treatment with cisplatin. All animals were sacrificed at 3 days after cisplatin injection. Representative images of H&E staining showed that the number of renal tubules with edema and structural damage were significantly reduced in the hucMSC-Ex group  were treated as described for the in vitro experiment. Mitochondrial membrane potential is considered a sensitive measurement of early apoptosis. Compared with the control group (NRK-52E cells without cisplatin), cisplatin reduced the mitochondrial membrane potential. hucMSC-Ex, but not HFL1-Ex, treatment antagonized the cisplatin-induced decrease in mitochondrial membrane potential Fig. . We alsoP < 0.05). The quantitative results of the integrated density of the Western blot image showed that pre-treatment with hucMSC-Ex increased the expression of autophagic maker proteins such as LC3B and Beclin-1 . Taken together, these results demonstrated that the activation of autophagy by hucMSC-Ex may contribute to its preventive effects on cisplatin-induced apoptosis and the inflammatory response in vitro.Considering the critical role of autophagy in cell apoptosis and tissue inflammation , 26, we In order to verify the activation of autophagy induced by the hucMSC-Ex in vivo model, 3MA (500 μg/kidney) and rapamycin (20 μg/kidney) were respectively injected under the kidney capsule before treatment with cisplatin. Representative images of H&E staining showed that the number of renal tubules with edema and structural damage were significantly reduced in the hucMSC-Ex group. However, co-injection with 3MA inhibited the protective effect of hucMSC-Ex on the renal structure. Rapamycin had a similar effect in preventing the structural damage of renal tubules to that of hucMSC-Ex Fig.  and b. FIncreasing evidence suggests that the administration of exogenous mesenchymal stem cells (MSCs) improves the recovery of injured tissue, including acute kidney injury (AKI) , 27–31. The role of autophagy as a degradative pathway is critical in regenerative medicine. Many reports show that basal or physiological autophagy contributes to the maintenance of cellular homeostasis and quality control of proteins and subcellular organelles. Under pathological conditions or cell stress, autophagy is induced which may serve as an adaptive and protective mechanism for cell survival. Autophagy is essential to the homeostasis and physiological function of podocytes in the kidney . NotablyIn order to prove whether hucMSC-Ex-mediated autophagy is essential for the inhibition of apoptosis and secretion of inflammatory cytokines, we pre-treated NRK-52E cells with the specific inhibitor of autophagy, 3-methyladenine (3MA). Inhibition of autophagy reduces the effects of hucMSC-Ex on anti-apoptosis and the inhibition of inflammatory cytokines. On the other hand, the autophagic inducer rapamycin has a similar effect on renal protection to that of hucMSC-Ex both in vitro and in vivo, suggesting that induction of autophagy is essential for the protective role of hucMSC-Ex in cisplatin-induced renal injury.From the above discussion, we propose a model as delineated in Fig. This study reveals that hucMSC-derived exosomes prevent against cisplatin-induced AKI through an autophagy-related mechanism. These findings provide a basis for the future use of exosomes as a new biological therapeutic approach for renal diseases and injuries.Additional file 1: Figure S1.scale bar = 50 μm). (C) Osteogenic differentiation was assayed by alkaline phosphatase staining . (PDF 270 kb)Characterization of hucMSCs. (A) Immunophenotyping of hucMSC. Histograms of MSCs stained with anti-CD13, CD44, CD29, CD90, CD105, HLA-1, CD45, HLA-DR, and CD34 shown with an overlaid isotype control. (B) Multi-lineage differentiation of hucMSCs. Adipogenic differentiation was assayed by Oil Red O staining . (C) Western blotting analyses of the expression of exosomal markers CD81, CD9, and CD63 in hucMSC-Ex. (D) TUNEL assay was performed to detect the apoptotic cells . (E) Electron microscope analyses of the expression of exosomal markers CD9 colloidal gold in hucMSC-Ex . (PDF 228 kb)Characterization of hucMSC-derived exosomes. (A) Nanoparticle tracking analysis on characteristics of the particles of hucMSC-Ex: (a) particle size and concentration, (b) the relative-intensity three-dimensional plot, (c) substantial shape of exosomes. (B) The morphology of hucMSC-Ex was observed under transmission electron microscopy hucMSC-Ex can activate autophagy in vitro. Quantitative RT-PCR analyses of ATG5 (A) and ATG7 (B) mRNA levels in NRK-52E cells. *Additional file 4: Figure S4.scale bar = 100 μm). (PDF 197 kb)hucMSC-Ex prevents secretion of inflammatory cytokines by activating autophagy in vivo. (A) Immunohistochemical analysis of TNF-α in kidney tissues (100×,


Multidrug resistance is a great obstacle in successful chemotherapy of colorectal cancer. However, the molecular mechanism underlying multidrug resistance is not fully understood. Dishevelled, a pivot in Wnt signaling, has been linked to cancer progression, while its role in chemoresistance remains unclear. Here, we found that Dishevelled1-3 was over-expressed in multidrug-resistant colorectal cancer cells (HCT-8/VCR) compared to their parental cells. Silencing Dishevelled1-3 resensitized HCT-8/VCR cells to multiple drugs including vincristine, 5-fluorouracil and oxaliplatin. Moreover, Dishevelled1-3 increased the protein levels of multidrug resistance protein 1 (P-gp/MDR1), multidrug resistance-associated protein 2 (MRP2), and breast cancer resistance protein (BCRP), Survivin and Bcl-2 which are correlated with multidrug resistance. shβ-catenin abolished Dishevelled-mediated these protein expressions. Unexpectedly, none of Dishevelled1-3 controlled β-catenin accumulation and nuclear translocation. Furthermore, the nuclear translocations of Dishevelled1-3 were promoted in HCT-8/VCR cells compared to HCT-8. Dishevelled1-3 bound to β-catenin in nucleus, and promoted nuclear complex formation and transcription activity of β-catenin/TCF. Taken together, Dishevelled1-3 contributed to multidrug resistance in colorectal cancer via activating Wnt/β-catenin signaling and inducing the expressions of P-gp, MRP2, BCRP, Survivin and Bcl-2, independently of β-catenin accumulation and nuclear translocation. Silencing Dishevelled1-3 resensitized multidrug-resistant colorectal cancer cells, providing a novel therapeutic target for successful chemotherapy of colorectal cancer. Colorectal cancer (CRC) is the third most prevalent malignancy and the fourth leading cause of cancer mortality worldwide . CurrentThe main mechanisms of MDR can be ranged into two principal types: “pump” and “non-pump” resistance , 9. The Wnt signaling plays an essential role in embryo development and adult tissue homeostasis, usually categorized as canonical and non-canonical signaling. The aberrant activation of canonical Wnt signaling (Wnt/β-catenin) triggers a wide variety of human pathologies such as various cancers . The travs 1.23 μM)  Figure , suggest) Figure . This suvs 6.53 μM) and oxaliplatin (31.15 μM vs 2.82 μM) compared with HCT-8 cells as well. However, silencing DVL using shDVL resensitized HCT-8/VCR cells to 5-FU and oxaliplatin. The IC50 of 5-FU (39.97 μM) in HCT-8/VCR cells was significantly decreased to 9.73, 11.72 and 8.90 μM by shDVL1, shDVL2 and shDVL3, respectively; and the IC50 of oxaliplatin (31.15 μM) was reduced to 5.96, 6.22 and 6.35 μM  assay showed that the binding of β-catenin to TCF-4 was increased in nucleus of HCT-8 cells transfected with pcDNA3.1-DVL1, -DVL2, or -DVL3 Figure , while dTo further understand how DVL stabilized nuclear β-catenin/TCF complex, we checked the nuclear translocation of DVL in HCT-8/VCR and HCT-8 cells. Here, we found that the protein levels of DVL1, DVL2 and DVL3 in cytoplasm and nucleus of HCT-8/VCR cells were higher than HCT-8 cells Figure . ImmunofCRC is a major cause of cancer-related death throughout the world. Significant improvements in survival rates of CRC patient have been achieved in recent years due to advances in cancer treatment. However, the resistance to chemotherapy agents usually occurs during advanced CRC treatment and facilitates cancer progression , 33. TheAlthough earlier studies have shown that DVL is relevant to tumor progression , 36, itsNext, we focused on the underlying mechanism that DVL regulated MDR in CRC. One of the major mechanisms of MDR is closely linked to ABC membrane transporters which can pump multiple chemotherapy agents out of cells to attenuate drugs-induced cytotoxicity, such as P-gp, BCRP and MRP , 39. HerPrevious studies have certified that P-gp, BCRP, MRP, Survivin and Bcl-2 are target genes of Wnt/β-catenin signaling –28. TherIt has been shown that DVL can increase the accumulation of β-catenin through inactivation of destruction complex containing APC, GSK3β and Axin, leading to translocation of β-catenin into nucleus , 46. HowPrevious reports have shown that DVL can increase β-catenin accumulation in cytoplasm to enhance translocation of β-catenin into nucleus, consequently promote nuclear complex formation of β-catenin/TCF and Wnt/β-catenin signaling , 55. HowIn conclusion, this study revealed that DVL contributed to MDR in CRC. All three DVL family members were over-expressed in multidrug-resistant CRC cells, where their nuclear translocations were increased. DVL1-3 bound to β-catenin in nucleus, acted as a coactivator to enhance the complex formation of β-catenin/TCF and its transcription activity Figure , inducinThe primary antibodies used in the present study: anti-DVL1, anti-DVL2, anti-DVL3, anti-β-catenin, anti-Flag, anti-HA, anti-Lamin B1 and anti-β-actin were obtained from Santa Cruz Biotechnology ; anti-TCF-4, anti-P-gp, anti-MRP2, anti-BCRP, anti-GSK3β, anti-p-GSK3β (Ser 9), anti-Survivin and anti-Bcl-2 were purchased from Cell Signaling Technology . The secondary antibodies were obtained from ZSGB-bio . shDVL1 (target sequence: 5′-GGAGGAGATCTTTGATGAC-3′), shDVL2 , shDVL3 (target sequence: 5′-GGAGGAGATCTCGGATGAC-3′) and shNC (negative control) were purchased from Genechem . cDNA encoding DVL1 was cloned into pcDNA3.1-Flag to construct DVL1 recombinant vector pcDNA3.1-Flag-DVL1. pcDNA3.1-HA-β-catenin was constructed in a similar manner. pcDNA3.1-Flag-DVL2 and pcDNA3.1-Flag-DVL3 were derived from pCMV5-3XFlag-DVL2 and XE251 pCDNA3.1(zeo)-hDsh3 which are gifts from Jeff Wrana (Addgene plasmid # 24802)  and RandThe human colorectal cancer cell line HCT-8 and its vincristine-resistant subline HCT-8/VCR were obtained from Shanghai Bogoo Biotechnology . The cells were authenticated by STR profile, and cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin and 100 units/ml penicillin at 37°C in a humidified 5% CO2 atmosphere. 0.5 μM vincristine sulfate was added into the medium to maintain drug-resistance phenotype of HCT-8/VCR cells.3/well) were seeded into each well of a 96-well plate. After incubation for 24 h, cells were treated with 100 μM DVL inhibitor 3289–8625  or transfected with shDVL for 24 h, followed by treatment with a range of concentrations of vincristine, 5-FU or oxaliplatin , for another 48 h. The cells were then washed twice using PBS, and 100 μl of 0.5 mg/ml MTT in medium culture was added to each well for 4 h incubation at 37°C. Following the culture medium was discarded, 150 μl DMSO was added to dissolve formazan blue. Absorbance at 490 nm was measured using an Ultra Microplate Reader . The inhibition ratio and IC50 (drug concentrations that achieved 50% growth inhibition) were calculated from the survival curves using the Bliss method.Drug cytotoxicity was evaluated using MTT assay. Briefly, cells  or 5-FU (12 μM) for additional 48 h. The cells were washed with cold PBS twice, gently resuspended in 100 μl binding buffer. The cell suspension was incubated with 1.5 μl Annexin V-FITC for 10 min at 4°C in the dark, and then 2.5 μl PI for 5 min. Apoptotic cells were quantified using a flow cytometer  and the CellQuest software. At least 10,000 cells were analyzed for each sample.Total cell proteins were obtained using RIPA lysis buffer. The cytoplasmic and nuclear proteins were extracted as described previously . ProteinCells were cultured on chamber slides to be 40–60% confluence, and then washed three times with phosphate buffer solution (PBS), fixed with 4% paraformaldehyde for 20 min, permeabilized using 0.2% Triton X-100 for 20 min, and blocked with 10% goat serum for 2 h at room temperature. Cells were then incubated with antibody against β-catenin or DVL (1:50 dilution) overnight at 4°C. The slides were washed three times with PBS, followed by incubation with Alexa Fluor 488-conjugated secondary antibodies (1:1000 dilution) for 2 h at room temperature. Nucleus was stained with DAPI (5 μg/ml) for 10 min and washed with PBS twice. The cells were visualized under a Zeiss confocal laser scanning microscope.4 cells per well and cultured to approximately 70% confluency. Then, pcDNA3.1-Flag-DVL and shDVL were respectively cotransfected with 0.2 μg of TOPflash plus 10 ng of pRL-SV40 or FOPflash plus 10 ng of pRL-SV40 by using Lipofectamine 3000 transfection reagent. After 48 h, the TOPflash and FOPflash luciferase activity were determined using a dual-luciferase reporter assay system . The luciferase activity of each sample was normalized against Renilla reporter pRL-SV40  luciferase activity for monitoring transfection efficiency.TOPflash and FOPflash constructs  are usually employed to assess β-catenin/TCF-dependent transcription . TOPflasBriefly, the protein extracts were incubated with 2 μg of anti-TCF-4, anti-β-catenin or anti-DVL antibody at 4°C overnight. Thereafter, protein A+G agarose beads were added into the mixture and incubated at 4°C for 6 h. The complexes were collected by centrifuging at 3,000 × g for 5 min at 4°C, and the precipitates were washed four times with ice-cold PBS. Bound proteins were separated from the agarose beads by boiling in sample buffer for 10 min, followed by western blotting analysis. The fusion proteins Flag-DVL and HA-β-catenin were immunoprecipitated and detected by incubation with anti-Flag and anti-HA antibody.t-test between two groups and by one-way ANOVA followed by Bonferroni test for multiple comparison involved. These analyses were performed using GraphPad Prism Software Version 5.0 , level of significance was set at a P < 0.05.Results were reported as mean ± standard deviation (SD) of three independent experiments unless otherwise specified. Data were analyzed by two-tailed unpaired Student's


Period 2-mutant mice (Per2m/m), which possess a circadian dysfunction, recapitulate the retinal vascular phenotype similar to diabetic retinopathy (DR). The vascular dysfunction in Per2m/m is associated with an increase in connective tissue growth factor (CTGF/CCN2). At the molecular level, CTGF gene expression is dependent on the canonical Wnt/β-catenin pathway. The nuclear binding of β-catenin to a transcription factor, lymphoid enhancer binding protein (Lef)/ T-cell factor (TCF/LEF), leads to downstream activation of CTGF. For this study, we hypothesized that the silencing of Per2 results in nuclear translocation and subsequent transactivation of the CTGF gene. To test this hypothesis, we performed immunofluorescence labeling for CTGF in retinal sections from wild-type (WT) and Per2m/m mice. Human retinal endothelial cells (HRECs) were transfected with siRNA for Per2, and the protein expression of CTGF and β-catenin was evaluated. The TCF/LEF luciferase reporter (TOPflash) assay was performed to validate the involvement of β-catenin in the activation of CTGF. Per2m/m retinas exhibited an increased CTGF immunostaining in ganglion cell layer and retinal endothelium. Silencing of Per2 using siRNA resulted in an upregulation of CTGF and β-catenin. The TOPflash assay revealed an increase in luminescence for HRECs transfected with Per2 siRNA. Our studies show that loss of Per2 results in an activation of CTGF via nuclear entry of β-catenin. Our study provides novel insight into the understanding of microvascular dysfunction in Per2m/m mice. Circadian rhythms play an important role in regulating many biochemical, physiological, and behavioral processes such as carbohydrate metabolism, sleep-wake cycle, and feeding. Disturbed circadian rhythms or circadian dyssynchrony is observed in shift work disorders and include, jet-lag, advanced sleep phase syndrome, and irregular sleep-wake rhythm . DiabetiThe core oscillator that drives the clock, the suprachiasmatic nucleus (SCN), is located in the hypothalamus; At the molecular level, circadian rhythms are orchestrated by interlocking autoregulatory transcription/translation feedback loops involving a set of clock genes . CompellPer2 and found that Per2m/m mice recapitulate a vascular phenotype of DR [Per2 is observed in a variety of human and mouse studies. In humans, a mutation in Per2 leads to a condition referred to as advanced sleep phase syndrome (ASPS), which is characterized by a short sleep cycle [Per2m/m mice possess a range of pathological disorders, such as cancer [Per2 dysregulation and the above disorders, specifically at the retina, is of intense interest to develop pharmacological targets.In a previous study, we demonstrated the critical role of clock regulatory gene pe of DR . The proep cycle , 16. Pers cancer , shortens cancer , and ends cancer , 20. ThePer2m/m retinas showed an increase in TGF-β1, CTGF, and fibronectin, suggesting an active involvement of the TGF-β1-CTGF pathway in vascular dysfunction of Per2m/mmice [CTGF is a secreted protein known to be involved in a variety of cellular events, such as angiogenesis, skeletogenesis, and wound healing . In the m/mmice . The Wntm/mmice . The nucm/mmice .Per2m/m retinas showed an increase in CTGF, the exact mechanism behind the upregulation of CTGF in Per2m/m retinas remains unknown. The purpose of this mechanistic study was to determine whether silencing of Per2 results in activation of the β-catenin-CTGF pathway and associated phenotypes. First, we performed scotopic electroretinogram (ERG) measurements and measured retinal thickness using the optical coherence tomography (OCT) in the retina of Per2m/m mice. Next, we measured CTGF expression in the retina from Per2m/m mice, and we assessed the effect(s) of Per2-silencing in HRECs on β-catenin and CTGF. Ultimately, our study highlights that the DR-like vascular phenotype observed in Per2m/m mice is due to a β-catenin mediated increase in CTGF expression.While our previous studies on Per2m/m female mice (B6.129-Per2tm1Drw/J) at 12 months of age were obtained as a gift from Dr. Choogon Lee, Florida State University, Tallahassee, FL). All animal studies were performed between 11 AM and 1 PM.All animal studies were performed in accordance with The Guiding Principles in the Care and Use of Animals (NIH) and the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. The protocol was approved by the institutional animal care and use committee of the University of Florida. The animal studies were completed while the authors were at the University of Florida, since then the research team has moved to the Indiana University School of Medicine. Wild type and For the ERG studies, the animals were dark-adapted overnight. The mice were anesthetized with a mixture of Ketamine and Xylazine. The pupils were dilated using 1% tropicamide and 2.5% phenylephrine. The platinum loop electrode  was placed over the cornea after the application of Gonak , and the scotopic ERG recordings were performed using an LKC NGIT-100 recording machine .The OCT assessments were performed on anesthetized mice using a Bioptigen Spectral Domain Ophthalmic Imaging System . Total retinal thickness was evaluated by manually placing of calipers extending from the retinal nerve fiber layer (RNFL) up to the retinal pigment epithelium (RPE), as described previously . The briHRECs were purchased from ATCC . Cells from passage two to seven were used for all experiments. Cells were cultured in pre-coated flasks/dishes/coverslips with attachment factor (AF), and M131 media was supplemented with microvascular growth factor (MVGS)  with 100U of antibiotic-antimycotic (Gibco-Life Technologies).Bandeiraea simplicifolia (BS)-1 isolectin  30 minutes prior to euthanasia. The eyeballs from mice were enucleated, fixed in 4% paraformaldehyde and embedded in paraffin. Sections were then deparaffinized and incubated in citrate plus buffer  for epitope retrieval [The retinal vessels were labeled with an intravenous injection of Rhodamine-conjugated etrieval . The retPer2 siRNA  using Lipofectamine RNAiMax reagent (Life Technologies) and were harvested after 24h for RNA analysis and protein analysis by Western blot. Cells transfected with negative control siRNA  were used as a control. siRNA-transfected cells were harvested between 11 AM and 1 PM and processed for protein or mRNA expression.HRECs were grown and transfected with 10μM Per2 (Hs 00256144), CTGF (Hs 00170014), β-catenin (CTNNB1 Hs 00355049), and β-actin (Hs 01060665) from Applied Biosystems.RNA was isolated using TRIzol (Life Technologies) as per the manufacturer’s protocol. One microgram of RNA was reverse transcribed using iScript cDNA synthesis kit . Gene-specific primers were used along with TaqMan Fast Universal master mix (Life Technologies), and respective mRNA levels were assessed using quantitative PCR . All genes were normalized to β-actin. Primers used were: Per2 siRNA-transfected cells were pelleted and lysed after 24 hrs in a RIPA buffer . Nuclear and cytoplasmic extracts were prepared using an NE-PER Nuclear and Cytoplasmic extraction kit  Protein concentration was estimated using the BCA assay , and equal amounts of proteins (50 μg) were loaded and separated on 4–12% Bis-Tris gels . Proteins were transferred to a PVDF membrane (Life Technologies), which was blocked with 5% milk or BSA as per the antibody requirement. The following antibodies were used for probing: CTGF , and β-catenin . The PVDF membranes were incubated with the aforementioned primary antibodies overnight at +4°C. The following set of antibodies were used as a secondary loading control: α-tubulin (1:800) and Histone-H3 . The secondary antibody incubations were performed for two hours at room temperature. The bands were visualized using ECL2 western blotting substrate (Pierce) on a XRS gel documentation system with Quantity One software .The Per2 or a control siRNA as per the manufacturer’s protocol. The cells were also co-transfected with a target gene luciferase reporter constructs and constitutively expressing Renilla construct. After 24h of transfection, the cells were harvested, and lysates were prepared and measured for luminescence using a Dual Luciferase Assay system  on a Synergy H1 Hybrid Reader . The ratio of luminescence for luciferase to Renilla was plotted for all experimental conditions.HRECs were co-transfected with a TCF/LEF construct  and a 10 μM The data were expressed as mean ± SEM. Statistical analysis was performed using a Graph Pad-Prizm 6  software. The statistical significance was evaluated using Student’s t-test or a Two-Way ANOVA followed by a Bonferroni’s post- hoc test. The data were considered to be statistically significant when the p-value was less than 0.05.Per2m/m mice, we performed scotopic ERG measurements and measured retinal thickness using the OCT. We found that the amplitude of ERG wave was reduced overall in the Per2m/m retinas . The HRECs were transfected with either siRNA  p<0.05;  and a 2. p<0.05;  mRNA levilencing .Per2-silencing would result in a dislodgement of β-catenin from the cytoplasm and its subsequent transfer to the nucleus, we transfected the HRECs with siRNA Per2 and stained with CTGF antibodies. The nuclear and cytoplasmic fractions of Per2 and control siRNA treated HRECs were separated. The Western blots were performed to determine the protein expression of β-catenin. The Histone H3 and α-tubulin were used as loading controls for nuclear and cytoplasmic fractions respectively. As shown in Per2 siRNA-treated HRECs showed an increase in β-catenin staining in a nuclear fraction as compared to the cytoplasmic fraction, suggesting nuclear entry of β-catenin due to Per2-silencing.To determine whether Per2 silencing results in a nuclear binding of TCF/LEF and downstream activation of CTGF. We co-transfected the HRECs with the siRNA Per2 and the TOPflash plasmid. We observed a 4-fold increase (p<0.05) in luciferase activity following Per2 silencing that was consistent with nuclear binding of TCF/LEF.  used in our studies does not produce detectable protein levels of PER2 [et al. (Per2tm1Brd) has the potential to generate a protein with a deletion of 87 amino acids [Our ERG and OCT findings differ from previous studies by Ait-Hymed et al. , which r of PER2 . Howeverno acids . Thus, wPer2m/m mice. The Per2m/m retinas exhibited an increased CTGF immunostaining in ganglion cell layer and retinal endothelium. We observed an upregulation of CTGF and β-catenin associated with Per2 silencing. CTGF is known to play a critical role in the pathogenesis of DR by promoting extracellular matrix (ECM) protein production [+/+) functional allele developed a full spectrum of the above features of DR. Taken together, this suggests that CTGF is involved in the pathological change of the retina in diabetes. Our study further ascertained an important role of CTGF in retinal vascular dysfunction due to lack of Per2.The predominant finding of our study is defining the critical role of CTGF in the development of retinopathy in oduction 25. In tPer2 is an important upstream regulator of the β-catenin-CTGF pathway in DR, and maintaining optimum levels of Per2 are necessary for protection from DR.Our studies show that clock function , β-catenin and CTGF are mechanistically interlinked, and this trio together might represent an important determinant in the pathogenesis of DR. For instance, previous studies suggest that hyperactivity of β-catenin is linked to pathological retinal neovascularization  and herePer2m/m mice [Per2m/m mice. Our studies using a TOPflash assay reveal that the silencing of Per2 results in a translocation of β-catenin from the cytoplasm to the nucleus and subsequent downstream activation of CTGF. These findings indicate a β-catenin-mediated increase in CTGF expression is responsible for the DR-like vascular phenotype observed in Per2m/m mice. Moreover, the β-catenin expression is increased following Per2-silencing in HRECs, in agreement with previous studies performed on colon carcinoma cells [We previously reported a loss of junctional integrity of VE-Cadherin in m/m mice . β-catenm/m mice . Howeverma cells . Our stuPer2m/m mice, CK1 shifts its enzymatic activity towards PER2, leading to its enhanced degradation and, therefore, reduced β-catenin phosphorylation. This, in turn, results in the nuclear entry of β-catenin, leading to downstream activation of targets such as CTGF.Another possible mechanism of nuclear translocation of β-catenin may be the result of the common phosphorylation and degradative pathway shared by both PER2 protein and β-catenin. The casein kinase 1 (CK1) acts as a key mediator that regulates nuclear import of β-catenin by virtue of its dual control over the phosphorylation of the PER2 and β-catenin. We speculate that in the Per2m/m mice by ascertaining the critical role of β-catenin-mediated CTGF increase. Further, our study paves a way for the development of pharmacological targets that, in the future, may curtail the development of DR.In conclusion, our study identified a novel pathway of retinopathy progression in


Wnt/β-catenin signaling is involved in patterning of bone primordia, but also plays an important role in the differentiation of chondrocytes and osteoblasts. During these processes the level of β-catenin must be tightly regulated. Excess β-catenin leads to conditions with increased bone mass, whereas loss of β-catenin is associated with osteoporosis or, in extreme cases, the absence of limbs. In this study, we examined skeletogenesis in mice, which retain only 25% of β-catenin. These embryos showed severe morphological abnormalities of which the lack of hindlimbs and misshaped front paws were the most striking. Surprisingly however, calcification of bone primordia occurred normally. Moreover, the Wnt-dependent regulatory network of transcription factors driving the differentiation of cartilage and bone, as well as the expression of extracellular matrix components, were preserved. These findings show that 25% β-catenin is insufficient for the correct patterning of bone primordia, but sufficient for their mineralization. Our approach helps to identify bone morphogenetic processes that can proceed normally even at low β-catenin levels, in contrast to those that require high β-catenin dosages. This information could be exploited to improve the treatment of bone diseases by fine-tuning the individual β-catenin dosage requirements. Summary: The correct allocation and patterning of skeletal elements during development requires a higher dosage of β-catenin than the maturation and mineralization of these primordia. Canonical Wnt signaling activates the transcription of β-catenin-dependent target genes during many stages of bone development and maintenance , wherebyEarly during embryonic development Wnt/β-catenin signaling plays crucial roles in determining the future body axes, and maintaining gastrulation and mesoderm production . During β-catenin only from the ROSA26 locus  or a gain of function (GOF) of different genes of the Wnt signaling pathway. GOF mutations in both mice and humans lead to unusually high bone mass , while L26 locus  in all t26 locus . We founCdx1::Cre mediates recombination in the three germ layers of the primitive streak region throughout the posterior embryo, caudal to the heart at mid-gastrulation (Cdx1::Cre)(β-catenin flox/flox)(ROSA26::β-catenin) mice, the endogenous β-catenin alleles are deleted and replaced by the ROSA26::β-catenin transgene. Homozygous expression of ROSA26::β-catenin leads to a β-catenin expression level of 25% compared to wild-type mice 18.5 knockdown embryos revealed that internal organs  were present, excluding a general defect during early gastrulation . Furthermore, we isolated DNA from these organs, as well as from brain, skin (head and abdomen), abdominal muscles and tail. Subsequent genotyping for endogenous β-catenin revealed that all tissues of foregut endodermal origin , anterior mesoderm-derived structures such as heart and spleen, and ectodermal organs like brain and skin from the head region still carried a floxed β-catenin allele . This means that Cre recombination had not taken place in these tissues. On the other hand, all posterior mesoderm-derived tissues  and skin isolated from the caudal half of the embryo lacked the floxed allele and only showed the deleted β-catenin gene product . Taken together, Cre-mediated recombination in our knockdown embryos took place in all mesodermal and ectodermal tissues that are derived from the posterior embryo at midgastrulation, including the fore- and hindlimb primordia. Phenotypically, mutant embryos showed major developmental defects with increasing severity in the caudal half of the embryo (hindlimbs and tail), as well as in the distal parts of the forelimbs . In more detail, most mutant embryos did not have any hindlimb structures  and only rudimentary pelvic bones . Forelimbs were always developed; however, their appearance was abnormal, and the digits showed severe malformations  and isolated forelimbs . Shape and size of the skeletal elements of forelimb zeugopodia and stylopodia seemed not to be altered between control . In rare cases, the forelimb zeugopodium consisted of only one bone . Conversely, the autopodia of β-catenin knockdown embryos always showed severe morphological alterations, with carpals, metacarpals and phalanges exhibiting irregular proportions and orientations . Hereby, the number of digits could be either increased  and in some cases fused phalanges were detected . Micro CT scans of control and mutant embryos  confirmed the findings of the skeletal staining. Moreover, in knockdown embryos, broadened, split lumbar vertebrae were found . Interestingly, there were only 11 instead of the expected 12 rib pairs present in the micro CT-scanned knockdown embryo .Expression of rulation . Thus, iype mice . These morelimbs ; Fig. S2ructures A,C and Gructures A,B, an urmations A. To gai control F and β-cncreased E or reduβ-catenin triggered us to analyze the expression of genes that are involved in the patterning of these structures. Two major signaling centers, the ZPA and the AER, control the morphogenesis of a limb. Hereby, Wnt3a stimulates the release of Fgf4 and Fgf8 from the AER. The Fgfs then induce the ZPA on the posterior end to produce Shh, which via Gremlin1 (Grem1) maintains Fgf expression in the AER. The Shh gradient further governs anterior-posterior patterning. Wnt7a and En1 secreted from the dorsal and ventral ectoderm, respectively, regulate dorsal-ventral patterning. We found in our analysis of E17.5 forelimb autopodia that the mRNA expression levels of all above mentioned genes were significantly reduced in β-catenin knockdown embryos except Wnt7a, whose expression was unchanged  or bone  and extracellular matrix proteins specific for cartilage  or bone . Cultures of primary rib chondrocytes derived from control or β-catenin knockdown embryos showed no morphological differences  despite the reduction of β-catenin (Ctnnb1) mRNA expression to 21% in knockdown cells  results from the autosomal recessive inactivation of the NT3 gene . In miceed tails . Furtherpromised . Howevered  morphogen gradient, originating from the zone of polarizing activity (ZPA), located at the posterior end of the growing limb  (β-catenin flox/flox) (ROSA26::β-catenin) (Cdx1::Cre)(β-catenin flox/flox)(ROSA26::β-catenin) mice, the endogenous β-catenin will be deleted in the caudal half of the embryo and replaced by the ROSA26 transgene. Mice that express β-catenin from both ROSA26 loci retain approximately 25% of β-catenin compared to wild types. In this study, embryos of both sexes were analyzed at E17.5 and E18.5. Genotyping of endogenous β-catenin was performed as previously described  Cre-recombinase under the control of the caudal type homeobox promoter (x1::Cre) ; (2) a cox/flox) ; (3) β-ccatenin) . In (Cdxescribed .For Hematoxylin-Eosin (HE) staining, paraffin embedded tissues were cut into 7 μm sections and rehydrated. Sections were then incubated in 1:10 dilution of Harris hematoxylin solution modified  for 3 min. After washing the slide in tap water for 10 min, they were incubated in Eosin Y solution alcoholic  for 2 min, followed by dehydration. Finally, the slides were mounted with Eukitt mounting solution . For von-Kossa staining for mineralized bone, the rehydrated sections were incubated in 1% silver nitrate  solution under UV-light for 3 h, rinsed in several changes of deionized water and incubated in 5% sodium thiosulfate  for 5 min to remove unreacted silver nitrate. After counter-staining with Nuclear Fast Red for 5 min, the dehydrated slides were mounted. For whole mount skeletal staining, Alcian Blue and Alzarin Red  staining was used as previously described . For qua5 cells/cm2. Before isolation of RNA some cells were stimulated with 100 ng/ml Wnt3a .Mouse rib chondrocytes were isolated from E18.5 embryos as described . Briefly® reagent according to the manufacturer's protocol. RNA concentration and quality was determined with a Nanodrop 1000 spectrophotometer  and 500-1000 ng were transcribed into cDNA using the Fast transcriptor cDNA synthesis kit . For subsequent steps the cDNA was diluted to 2 ng/µl. All qRT-PCR reactions were run in triplicate using the Universal Probe Library system from Roche. 9 µl cDNA and the appropriate primers and probes (see Table S1) were used for each reaction.Total RNA was isolated with TRIzolt-tests. P-values <0.05 were considered statistically significant.The results of qRT-PCR and von-Kossa stained area were tested for significance using unpaired, two-tailed Student's