LRRK2 mutations and risk variants increase susceptibility to inherited and idiopathic Parkinson’s disease, while recent studies have identified potential protective variants. This, and the fact that LRRK2 mutation carriers develop symptoms and brain pathology almost indistinguishable from idiopathic Parkinson’s disease, has led to enormous interest in this protein. LRRK2 has been implicated in a range of cellular events, but key among them is canonical Wnt signalling, which results in increased levels of transcriptionally active β-catenin. This pathway is critical for the development and survival of the midbrain dopaminergic neurones typically lost in Parkinson’s disease.Lrrk2 knockout mice and fibroblasts to investigate the effect of loss of Lrrk2 on canonical Wnt signalling in vitro and in vivo. Micro-computed tomography was used to study predicted tibial strength, while pulldown assays were employed to measure brain β-catenin levels. A combination of luciferase assays, immunofluorescence and co-immunoprecipitation were performed to measure canonical Wnt activity and investigate the relationship between LRRK2 and β-catenin. TOPflash assays are also used to study the effects of LRRK2 kinase inhibition and pathogenic and protective LRRK2 mutations on Wnt signalling. Data were tested by Analysis of Variance.Here we use Lrrk2 knockout cells display increased TOPflash and Axin2 promoter activities, both basally and following Wnt activation. Consistently, over-expressed LRRK2 was found to bind β-catenin and repress TOPflash activation. Some pathogenic LRRK2 mutations and risk variants further suppressed TOPflash, whereas the protective R1398H variant increased Wnt signalling activity. LRRK2 kinase inhibitors affected canonical Wnt signalling differently due to off-targeting; however, specific LRRK2 inhibition reduced canonical Wnt signalling similarly to pathogenic mutations.Loss of Lrrk2 causes a dose-dependent increase in the levels of transcriptionally active β-catenin in the brain, and alters tibial bone architecture, decreasing the predicted risk of fracture. . In agreement, over-expressed LRRK2 binds and represses β-catenin, suggesting LRRK2 may act as part of the β-catenin destruction complex. Since some pathogenic LRRK2 mutations enhance this effect while the protective R1398H variant relieves it, our data strengthen the notion that decreased canonical Wnt activity is central to Parkinson’s disease pathogenesis.Loss of LRRK2 causes increased canonical Wnt activity in vitro and in vivoThe online version of this article (doi:10.1186/s13024-017-0153-4) contains supplementary material, which is available to authorized users. PD is the second most common neurodegenerative disease worldwide  using a dounce homogenizer and then clarified by centrifugation at 14 000 g for 10 min. Samples for Western blotting were denatured by the addition of 4× LDS sample loading buffer and 10× sample reducing agent (both Invitrogen) followed by heating to 99 °C for 10 min. Samples for ECT pulldown experiments were assayed for protein concentration by bicinchoninic acid (BCA) assay (Pierce).Brains taken from 22 to 26 week old male or 60–64 week old female mice were rinsed in phosphate-buffered saline (PBS) and immediately transferred to ice. All subsequent steps were performed at 4 °C. Brains were homogenized into 5 ml brain lysis buffer  and subjected to at least three freeze-thaw cycles. Extracts were clarified by centrifugation and transferred to a fresh tube, with a small aliquot used to verify protein expression by Coomassie staining. In parallel, 1 ml of Glutathione Sepharose 4 Fast Flow beads  was washed in 5 ml bacterial lysis buffer. To generate GST-ECT beads, the beads were added to cleared bacterial lysate and incubated for 30 min at 4 °C on a rotator. Beads were washed five times in bacterial lysis buffer and resuspended in the same buffer supplemented with 50% glycerol, prior to storage at −20 °C. GST-ECT beads were equilibrated into brain lysis buffer before use in ECT pulldown experiments.To induce expression of GST-ECT protein, BL21 pcDNA3-FLAG-β-catenin , Axin2-v/v) foetal bovine serum, 2 mM glutamine, 100 U/ml penicillin G and 100 μg/ml streptomycin at 37 °C in 95% air-5% CO2. Cells for immunofluorescence were seeded onto poly-d-lysine-coated coverslips. In all cases cells were transfected with Fugene HD reagent (Roche) and harvested 24 h post-transfection.HEK293 cells (ATCC CRL1573) and LRRK2 knockout and wild-type control mouse embryonic fibroblast cells  were grov/v).The LRRK2 kinase inhibitors LRRK2-in-1 , TAE684 v/v) Triton X-100, plus 1X complete protease inhibitor cocktail (Roche) and 1X Halt phosphatase inhibitor cocktail. Following centrifugation  1 ml of cell lysate was added to 40 μl of anti-FLAG M2 affinity gel (Sigma) and incubated for 1 h at 4 °C on a turning disk. The affinity gel was washed four times in extraction buffer and FLAG fusion proteins eluted with 150 ng of 3 × FLAG peptide (Sigma) for 30 min at room temperature. Eluates were denatured by the addition of 4× LDS sample loading buffer and 10× sample reducing agent (both Invitrogen) followed by heating to 99 °C for 10 min.Cells were grown in 10 cm plates and transfected with 4 μg of the relevant plasmids. Twenty-four hours later cells were lysed in a solution containing 150 mM NaCl, 50 mM Tris (pH 7.5), 2 mM EDTA (pH 8), 1% (w/v) BisTris pre-cast gels (Invitrogen). Proteins were transferred to polyvinylidine fluoride (PVDF) membranes (Millipore), rinsed briefly in Tris-buffered saline plus 0.1% (v/v) Tween 20 (TBS-T), and blocked by incubation in TBS-T containing 5% (w/v) non-fat dry milk (blocking buffer) for 30 min. Primary antibody incubations were performed in blocking buffer overnight at 4 °C. Antibody concentrations used were: Anti-myc (Sigma), 1:2000 dilution; anti-FLAG (Sigma), 1:2000 dilution; anti-β-catenin , 1:2000; anti-Calnexin (AbCam), 1:3000 dilution; rabbit anti-LRRK2 (Epitomics ‘MJFF2’), 1:1000; and mouse anti-LRRK2 (NeuroMab ‘N138/6’), 1:1000. The following morning, membranes were washed three times in TBS-T, prior to incubation in HRP-conjugated secondary antibody (Santa Cruz) at 1:2000 in blocking buffer for 1 h at room temperature. After an additional three washes, staining was visualized using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Chemiluminescent Substrate (both Pierce) and a Syngene GeneGnome Imaging system. Images were quantified using GeneQuant software.Protein samples were loaded onto 4–12%  paraformaldehyde and stained with antibodies to myc and FLAG (Sigma) as described previously [Cells were transfected with 500 ng of the relevant plasmid per coverslip. 24 h post-transfection, cells were treated with 10 μM MG132 for 1 h to allow accumulation of β-catenin. Cells were fixed in 4%  on normally distributed trabecular bone data using GraphPad Prism 6 . For cortical bone, analysis graphs were developed using the programming language “R”, version 3.1.1 . Data distribution’s normality was determined using the Shapiro-Wilk test and the Kolmogorov-Smirnov goodness of fit test. A single factor ANOVA was used to analyse equality of means between groups. Group means were compared using the Tukey-Kramer method.One way analysis of variance (ANOVA) was used to determine the effects of genotype  parameter, with lower values obtained at a proximal region, but significantly higher values at two distal regions , trabecular bone volume (BV), BV/TV, trabecular number and thickness  but significant increase in basal TOPflash activity compared to wild-type cells . Indeed, in the case of FLAG-β-catenin transfection, the activation of canonical Wnt activity was over 30 times higher than in wild-type cells . Thus, in addition to causing an increase in total β-catenin in the brains of younger male Lrrk2 knockout mice, loss of Lrrk2 causes a dose-dependent elevation of free β-catenin in the brains of both younger males and aged females, albeit to different extents. Taken together, these data provide strong evidence for elevated canonical Wnt signalling in the brains of Lrrk2 deficient animals.ECT assays performed on brain extracts from a set of male rk2 Fig. . The difWe previously reported that the LRRK2 kinase inhibitor LRRK2-in-1  represseIn agreement with previous data, 1 μM LRRK2-in-1 produced a robust inhibition of DVL1-driven TOPflash activity . Thus, and lung –93. Intrand lung , and alsand lung . As suchLrrk2 null mice is particularly interesting to consider in light of the growing links between Parkinson’s disease and osteoporosis. It has long been known that PD patients are at increased risk of bone fractures, although this has traditionally been accounted for by the greater risk of falling due to postural instability, together with less exercise creating more fragile bones directly and indirectly via less exposure to sunlight (leading to lower Vitamin D). However, recent advances suggest osteoporosis and PD are more intimately linked, with rates of co-morbidity far higher than assumed [Lrrk2 mutations, and we are unaware of any similar studies on human LRRK2 patients or unaffected carriers. However, our data suggest strongly that these individuals have lowered canonical Wnt activity (Fig. Finally, increased predicted bone strength in  assumed . In fact assumed . A recen assumed . As suchLrrk2 deficient mice display elevated canonical Wnt signalling and increased indices of predicted bone strength. Four out of seven pathogenic LRRK2 mutations are associated with statistically significant increases in repression of Wnt activity, whilst a protective mutation has the opposite effect. Thus, this study supports the connection between decreased Wnt signalling and Parkinson’s disease, and suggests that the deregulation of this pathway may not only contribute to neurodegeneration, but may also account for the increased incidence of osteoporosis seen in PD patients.Our data indicate that LRRK2 is a repressor of β-catenin and therefore


Musa acuminata cv. “Grande Naine” (AAA) and Musa acuminata × balbisiana Colla cv. “Bluggoe” (ABB)—when these were fed on by the banana weevil (Cosmopolites sordidus (Germ.) (Coleoptera: Curculionidae)) and the banana stem weevil (Odoiporus longicollis (Oliver) (Coleoptera: Curculionidae)). The chemical constituents of the banana material were separated by means of chromatographic techniques and identified by NMR spectroscopy. One new compound, 2-methoxy-4-phenylphenalen-1-one, was found exclusively in the corm material of “Bluggoe” that had been fed on by the weevils.Phenylphenalenone-type compounds accumulated in the tissues of two banana cultivars— Musa spp.) are a major staple food for more than 400 million people in developing countries in the tropics and subtropics, and an important export product for the Philippines and many Latin American countries + .


TNF-α, IL-8, VEGF, and uPA. In addition, the α-catenin SUMOylation mutant shows impaired suppression of tumor growth. These results demonstrate that SUMOylation at lysine 870 of α-catenin plays a key role in the suppression of the NF-κB pathway, which inhibits breast cancer tumor growth and migration.α-catenin has been demonstrated to suppress several different types of cancers. Here we demonstrate that α-catenin is modified by SUMO protein, which covalently binds α-catenin at the carboxy terminus at lysine 870. Substitution of lysine 870 with arginine completely abolishes α-catenin SUMOylation. This modification can be removed by SENP1. However, α-catenin SUMOylation does not affect its stability and subcellular localization. In addition, we observed that the SUMOylation-deficient α-catenin mutant has a reduced interaction with IκBα which prevents subsequent ubiquitination of IκBα, and therefore a reduced suppression of expression of the NF-κB target genes  CTNNA1 (encoding α-catenin) is one of the most frequently deleted2 or mutated3 genes in cancer. α-catenin is an essential protein in adherens junctions, which are critical for maintaining intercellular adhesion and cellular polarity5. The tumor-suppressor activities of α-catenin involve following signaling pathways. An siRNA screen suggested that the YAP1 may be a downstream effector of α-catenin6, which inhibits the transcriptional activity of YAP1 by promoting its cytoplasmic sequestration7. α-catenin is frequently downregulated in human skin cancer8, and an α-catenin-knockout skin squamous cell carcinoma mouse model showed upregulation of many NF-κB downstream genes9. α-catenin suppresses p50/p65 nuclear localization by binding to IκBα and increasing its protein stability in breast cancer10. Moreover, α-catenin directly interacts and promotes β-catenin degradation11.The tumor-suppressor gene 12. SUMOylation regulates numerous cellular stress responses including DNA repair, transcriptional regulation, and RNA metabolism13. Like with ubiquitination, SUMOylation involves several steps and three classes of enzymes including E1 enzyme SAE1/SAE2, only E2 enzyme Ubc9 and diverse E3 enzymes which accelerate the SUMOylation rate of final step14. SUMOylation is a dynamic process which regulates substrate functions including subcellular distribution, protein stability and control of protein interactions16. Numerous studies indicate that NF-κB pathway, such as stabilizing IκBα17, activation of the IκB kinase NEMO18, and promoting p50 maturation19, is regulated by SUMOylation. Given that α-catenin can interact with Ubc920 indicates that α-catenin is a target of SUMO. A computer analysis of the α-catenin protein sequence reveals two conserved SUMOylation sites: lysine 86 and 870. In this article, we demonstrate that α-catenin is SUMOylated at lysine 870 in vivo. Mutating K870 to arginine reduces the binding of α-catenin to IκBα and negatively regulates NF-κB target gene expression, therefore inhibiting α-catenin tumor-suppressor activity in breast cancer. Collectively, our findings suggest that α-catenin SUMOylation is of great importance in regulating α-catenin tumor-suppressor activity.SUMOylation is a post-translational modification where SUMO covalently binds to target proteins21. To further confirm this, HEK293T cells were transfected with Flag-α-catenin together with WT SUMO1 or its mutant SUMO1-G97A and analyzed the SUMOylated proteins. We observed α-catenin was only modified by WT SUMO1 but not SUMO-G97A  Fig. . We consnin Fig. . These dSince SUMOylation usually regulates substrate subcellular distribution, we wondered whether α-catenin SUMOylation is required for localizations to adherens junctions by transfecting WT Flag-α-catenin or the K870R mutant into MDA-MB-231 cells and monitoring α-catenin subcellular localization using immunofluorescent staining. The results showed that α-catenin mainly aggregated in the cytoplasm, regardless of mutation status Fig. . To conf24, Hippo-Yap7 and NF-κB signaling10 pathways. To determine whether SUMOylation of α-catenin regulates these pathways, we performed TOPFlash, Tead4 luciferase, and pNFκB-luciferase assays in HEK293T cells, respectively. Unexpectedly, the K870R α-catenin mutant showed significantly reduced inhibition of the pNF-κB-luciferase with or without TNF-α stimulation, whereas the α-catenin SUMOylation mutant did not affect TOPFlash and Tead4 luciferase activity compared to WT α-catenin  is important for mediating protein-protein interactions46, we speculate that IκBα contains a SIM motif which promotes the interaction between IκBα and SUMOylated α-catenin.Since no suitable therapy and drugs for triple-negative breast cancer cells (TNBC), it has a lower five-year life rate than other subtypes47D Fig. . ActivatIn this research, we demonstrate that α-catenin is SUMOylated at lysine 870, enhancing its interaction with IκBα, and therefore leading to upregulation of IκBα protein levels and inactivation of NF-κB pathway. We also demonstrate that α-catenin SUMOylation does not affect protein stability Fig.  and subc48. Tead4 luciferase construct was a kind gift from Dr. Ximei Wu49. The following plasmids were a kind gift provided by Dr. Hai-long Piao: SFB-α-catenin construct, SFB IκBα construct, Myc-α-catenin construct, pLOC α-catenin construct, Myc-IκBα construct, HA-Ub (ubiquitin) construct, pNF-κB luciferase reporter construct, TOPFlash luciferase reporter construct, pGPIZ CTNNA1 shRNA construct10. GFP-α-catenin was amplified from SFB α-catenin construct. GFP-α-catenin sense primer: 5′-CGGAATCCACTGCTGTCCATGCA-3′. GFP-α-catenin reverse primer: 5′-GGGGTACCCCTTAGATGCTGTCCATAGCTT-3′ pLOC α-catenin mutant (K870R), GFP α-catenin mutant (K870R) and Flag-α-catenin mutants  were generated using a site-directed mutagenesis kit .Myc-labeled SUMO1, SUMO2, SUMO3, Flag-SENP1, Flag-SENP1m, GFP-SUMO1, and GFP-SUMO1-G97A were previously discribedThe following antibodies were used: Flag, GFP, HA, Myc, α-catenin rabbit polyconal antibodies and Flag, GAPDH mouse monoclonal antibodies , rabbit anti-IκBα antibody , rabbit SUMO1 polyclonal antibody , SUMO1, Fibrillarin mouse monoclonal antibodies and rabbit anti-UBC9 , mouse anti-TNF-α, rabbit anti-uPA, rabbit anti-Ki-67 antibodies , mouse anti-β-tubulin antibody .50.Transfection assay was performed with Lipofectamine 2000 .MD-MBA-157 cells were purchased from China Infrastructure of Cell Line Resource and cultured under conditions specified by the provider. HEK293T, MCF-7, T47D, MD-MBA-231, and 4T1 cells were previously describedLentivirus containing medium was generated using three plasmids lentivirus package system. Cells were incubated with virus and selected using suitable antibiotic.51. MDA-MB-231 cells (2 × 104) or MDA-MB-157 cells (5 × 104) were infected with virus and selected by blasticidin before plated.Transwell assay was previously described51.Cells were plated into a 24-well plate at a confluency of 30%, and transfected with relevant plasmids 24 h later. The luciferase activity was tested as previously described32. TNE lysis buffer  containing 20 mM N-Ethylmaleimide  for exogenous SUMOylation assay. The method for analysis of endogenous α-catenin-SUMO1 was previously described17. Immunohistochemistry (IHC) were performed as previously described51.Cells extracts were generated using TNE lysis buffer. Western blot and Co-immunoprecipitation were previously describedCytoplasm and nucleus separation assay was performed using Protein Extraction Kit (Wanleibio).51.MDA-MB-231 cells were transfected with Flag-α-catenin or its SUMOylation mutant K870R using Lipofectamine 2000. Transfected cells were seeded at coverslip 48 h later. Immunofluorescence staining was described in our previously studies32.For crystal violet staining assay, cells were infected with virus containing medium, and then plated in 35 mm dish. After clones were clearly visible, cells were stained with crystal violet. MTT assay was described in our previous studyTotal RNA was extracted using RNAiso reagent . cDNA was generated using a Reverse Transcription System (Takara). Normalized abundance of mRNA were measured using SYBR premix Ex Taq (Takara). The primers were designed as follows: TNF-α sense primer: cccaggcagtcagatcatcttc. TNF-α reverse primer: agctgcccctcagcttga. IL-8 sense primer: 5’-ttttgccaaggagtgctaaaga IL-8 reverse primer: aaccctctgcacccagttttc. VEGFA sense primer: cgggaaccagatctctcacc. VEGFA reverse primer aaaatggcgaatccaattcc. uPA sense primer: agtgtcagcagccccact. uPA reverse primer ccccctgagtctccctgg. CCND1 sense primer: gctgctcctggtgaacaagc. CCND1 reverse primer aagtgttcaatgaaatcgtgcg. C-MYC sense primer: cagctggagatgatgaccga. C-MYC reverse primer ttgatgaaggtctcgtcgtc. CTGF sense primer: gcccagacccaactatgattag. CTGF reverse primer: tctccgtacatcttcctgtagt. CRY61 sense primer: gactgtgaagatgcggttcc. CRY61 reverse primer ctgtagaagggaaacgctgc.10.BALB/c mice (8 weeks old) were purchased from the Laboratory Animal Center of Dalian Medical University. Mice were managed and raised according to the procedures of the Ethics Committee for Biology and Medical Science of Dalian University of Technology. BALB/c mice were implanted subcutaneously with 4T1 cells infected with relevant virus. Tumor size and volume were recorded once a week. Tumor volume calculating method was used according to a previous studyt-test was used when the results from two groups were compared. Experimental data is considered to be significant when p < 0.5. *p < 0.05, **p < 0.01.Data were showed as means ± SDs. An unpaired Supplementary figure legendSupplementary Figure. 1Supplementary Figure.2Supplementary Figure.3Supplementary Figure. 4


As a result, it contributes to carcinogenesis and tumor progression of several cancers, including colon cancer, hepatocellular carcinoma, pancreatic cancer, lung cancer and ovarian cancer. β-Catenin is a pivotal component of the Wnt signaling pathway and it is tightly regulated at three hierarchical levels: protein stability, subcellular localization and transcriptional activity. Uncovering the regulatory mechanisms of β-catenin will provide new insights into the pathogenesis of cancer and other diseases, as well as new therapeutic strategies against these diseases. In this review we dissect the concrete regulatory mechanisms of β-catenin from three aspects mentioned above. Then we focus on the role of β-catenin in cancer initiation, progression, dormancy, immunity and cancer stem cell maintenance. At last, we summarize the recent progress in the development of agents for the pharmacological modulation of β-catenin activity in cancer therapy.Wnt/β-catenin signaling is an evolutionarily conserved and versatile pathway that is known to be involved in embryonic development, tissue homeostasis and a wide variety of human diseases. Aberrant activation of this pathway gives rise to the accumulation of β-catenin in the nucleus and promotes the transcription of many oncogenes such as  Jun, c-Myc and CyclinD-1 in a tissue specific manner, most of which encode oncoproteins. In more than half of all cancer cases, such as colorectal carcinoma, breast cancer, liver carcinoma, melanoma and leukemia, β-catenin accumulates within the nucleus or cytoplasm [β-Catenin is a multifunctional protein with a central role in physiological homeostasis. Its aberrant high expression leads to various diseases including cancer. β-Catenin is the mammalian homologue of the drosophila armadillo gene. It acts both as a transcriptional co-regulator and an adaptor protein for intracellular adhesion. Wnt is the chief regulator of β-catenin, which is a family of 19 glycoproteins to regulate both the β-catenin-dependent  and -independent  signaling pathways –5. In thytoplasm –10. It hytoplasm . Besidesytoplasm . The actThe stabilization of β-catenin is critical in tumorigenesis, which is usually induced by aberrant Wnt activation and somatic gene mutations of β-catenin or destruction complex components , 14. WitIn the absence of Wnt, β-catenin is delivered to proteasome for degradation by the so-called cytoplasmic “destruction complex”, which is composed of APC, Axin , GSK3β, and casein kinase 1α (CK1α) . APC recA central question in Wnt-signaling transduction is the regulation of β-catenin phosphorylation, in which kinases and phosphatases jointly take part. Thus, a balance between phosphorylation and dephosphorylation must be reached. PP2A is a heterotrimeric complex, in which PR55α (a regulatory subunit) suppresses the function of GSK3β by dephosphorylating β-catenin . PhosphoDestruction complex formation is a required step for β-catenin phosphorylation. LRP6, frequently rearranged in advanced T-cell 1 (FRAT1) and aldolase are proteins involved in the regulation of destruction complex formation. As a receptor of Wnt, LRP6 acts as an inhibitor of GSK3β in response to Wnt stimulation . Upon acProtein kinase Cζ (PKCζ) is another protein kinase that phosphorylates Wnt/β-catenin signaling through direct phosphorylation of β-catenin at Ser45 in a CK1α-independent manner and promoting β-catenin degradation. This effect represses intestinal stem cell function and tumorigenesis . DepletiCyclinD1 gene transcription, and promoting the development of HCC [Some proteins indirectly regulate β-catenin phosphorylation and degradation through modulating GSK3β kinase activity. Dachshund inactivates Wnt signaling and inhibits hepatocellular carcinoma (HCC) growth through decreasing GSK3β Ser9 phosphorylation . The latt of HCC .The ubiquitination of β-catenin is a complex process, which is either phosphorylation dependent or independent. Also, this post-translational modification happens either in the cytoplasm or in the nucleus Figure . ActuallIn the Wnt-on state, β-catenin translocates into the nucleus due to the disassembly of the β-catenin destruction complex. Recently, researchers found that β-catenin ubiquitination in the nucleus are mainly mediated by c-Cbl and tripartite motif-containing protein 33 (TRIM33). C-Cbl is a Ring finger ubiquitin E3 ligase expressed in endothelial cells, which binds to the nuclear active non-phosphorylated β-catenin via the armadillo-repeat region. Indeed, c-Cbl also mediates unbiquitination of cytoplasmic β-catenin in a similar way to Jade1 . TRIM33 Tribbles homolog 2 (TRIB2), a member of Tribbles protein family, is found to be a direct target gene of Wnt/β-catenin and promotes HCC cell survival and transformation. Interestingly, a negative feedback loop exists between TRIB2 and Wnt signaling whereby TRIB2 facilitates the ubiquitination of TCF4 and β-catenin . β-TrCP,Just like kinases and phosphatases oppose each other in the regulation of phosphorylation, there are also deubiquitinases that oppose ubiquitin ligases in regulating β-catenin ubiquitination. Ubiquitin-specific peptidase 7 (USP7) mediates deubiquitination of β-catenin and enhances canonical Wnt signaling . ThroughUnlike phosphorylated sites Ser33, Ser37 and Thr41, phosphorylation of some sites in β-catenin helps maintaining its stability. For instance, phosphorylation of β-catenin on Ser675 by PKA enhances β-catenin stability through inhibiting its ubiquitination , 50. MorAutophagy is an evolutionarily highly conserved catabolic pathway that degrades cellular macromolecules and organelles. Recent studies indicate that there exists a crosstalk between Wnt/β-catenin signaling and autophagy. On one side, the Wnt/β-catenin signaling pathway acts as a negative regulator of autophagy. Under nutrient rich conditions, autophagy is maintained at basal level only. β-Catenin limits autophagosome formation and functions as a transcriptional co-repressor of p62 through binding to the transcription factor TCF4 . In multNuclear concentration of β-catenin is the hallmark of Wnt/β-catenin signaling. As a consequence of aberrant activation of Wnt signaling, the active form of β-catenin translocates into the nucleus to act as a transcriptional activator. There exist specific regulatory mechanisms governing the subcellular translocation of β-catenin, independent of its stability.The nuclear envelope is a double membrane permeated by nuclear pores. Proteins larger than 50 kDa with the nuclear localization sequence (NLS) or the nuclear export sequence (NES) require active transport by transport receptors (including importins and exportins) via the nuclear pore complexes (NPC), which are composed of 30-50 different nucleoporin proteins. Fagotto et al. demonstrated that although β-catenin does not contain NLS, it still could be imported into the nucleus by directly binding to the nuclear pore machinery. The characteristics of β-catenin are similar to importin-β, whose nuclear transport does not require additional components of the transport machinery, such as importins, exportins or RAN-GTPase . This fuExcept for direct interaction with NPC, many nuclear import or export chaperones mediate the subcellular translocation and nuclear retention of β-catenin  Figure . Smad3/4In addition to the chaperones that directly interact with β-catenin and assist in its translocation, some oncoproteins or tumor suppressors regulate the shuttling process in an indirect way. N-myc downstream-regulated gene 1 (NDRG1), a potent metastasis suppressor, inhibits EMT in prostate and colon cancer cells. Overexpression of NDRG1 in colon cancer cells inhibits the nuclear localization of PAK4, increasing the levels of membrane-associated β-catenin and downregulating β-catenin transcriptional activity . Custos In general, post-translational modification of β-catenin itself or in coordination with the import or export chaperones directly regulate the subcellular localization of β-catenin. In addition, many upstream regulators of those chaperones also influence the nuclear translocation of β-catenin. Besides, some proteins promote β-catenin retention in the nucleus or cytoplasm through a direct protein–protein interaction manner. Most of the regulators play critical roles in tumorigenesis and tumor development, providing new targets for cancer treatment.As a protein with no DNA binding ability, β-catenin is thought to form a transcriptional complex with TCF/LEF, p300/CBP, and other proteins. It functions as a transcriptional activator following nuclear translocation. Studies show that the complex is diverse and complicated in different types of cancers. More and more co-activators and regulators have been found, which involve and influence Wnt/β-catenin transcriptional activity.TCF proteins have DNA-binding domains, but no intrinsic transcriptional activity. In the absence of β-catenin, TCF4 interacts with Groucho/TLE, CtBP and histone deacetylase proteins, which leads to promoter repression. Once monoubiquitylated by X-linked inhibitor of apoptosis (XIAP), Gro/TLE dissociates from TCF/Lef, thereby allowing β-catenin-TCF/Lef complex assembly and promoting Wnt-specific transcriptional program . In the CyclinD1 gene expression [legless (the homolog of human BCL9) and pygopus are positive regulators of the Wnt/β-catenin signaling pathway [c-Myc and CyclinD 1. SOX10 is a member of the SOX protein family and its expression is upregulated in HCC. Interestingly, SOX10 expression is positively correlated with elevated β-catenin levels in HCC, and β-catenin activity promotes oncogenic effects of SOX10. SOX10, TCF4 and β-catenin form a ternary complex, which binds to similar DNA sequences as TCF4 via a single HMG domain, and facilitates the binding of TCF4 with β-catenin [Homologues p300 and CBP are histone acetyltransferases (HATs), which recruit basal transcriptional machinery to the promoters. The C-terminus of β-catenin interacts directly with the CH3 domain of p300, thus alleviating the repression of Wnt-target genes induced by Groucho/TLE and so on . Furtherpression , 66. Thepression . While,  pathway . BCL9 ac pathway . The leu pathway . p68 and pathway . Except -catenin . β-TrCP1-catenin . Hence, The co-activators above either have chromatin remodeling capacities, transcriptional activities or have direct DNA-binding abilities. Besides, they all interact with β-catenin or other components to form a complex and enhance its transcriptional activity. In addition, some regulators directly bind to β-catenin, interrupting or enhancing its interaction with other components of the complex. Tumor adaptation to hypoxia is mainly mediated by two transcription factors, the hypoxia-inducible factor 1α or 2α (HIF1α or HIF2α). HIF1α competes with TCF4 for direct binding to β-catenin at the promoter region of HIF1α target genes. β-Catenin enhances HIF1α-mediated transcription, thereby promoting cell-cycle arrest, cell survival and adaptation to hypoxia . Like HIMYC [Many proteins interact with components of the transcriptional complex to regulate β-catenin transcriptional activity. Here, we focus on the regulators of TCF4 and p300/CBP. The TCF4-related regulators include HOXB13, SOX4, RUNX3, CDK8, TCTP and Daxx. HOXB13 is exclusively overexpressed in the normal prostate and the colorectum tissues. HOXB13 is a negative regulator of TCF4 that promotes TCF4 degradation. A lower HOXB13 expression in colorectal cancer confers TCF4-mediated transactivation . On the MYC . TranscrMYC . TogetheMYC . Daxx acMYC . The molp300/CBP-related regulators include HIF2α, KLF4 and Smad2. As mentioned above, HIF2α activates β-catenin by recruiting p300 through its transactivation domains. KLF4 is a transcription factor highly expressed in the adult intestine. In contrast to HIF2α, KLF4 was demonstrated to directly interact with the C-terminal transactivation domain of β-catenin and block p300/CBP recruitment by β-catenin. KLF4 inhibits p300/CBP-mediated β-catenin acetylation as well as histone acetylation on Wnt target genes, resulting in the inhibition of Wnt signaling . SMAD2, Associations between Wnt/β-catenin signaling and cancer initiation, tumor growth, tumor metastasis, tumor dormancy, tumor immunity and tumor stem cell maintenance have been revealed over the last two decades. Constitutive activation of Wnt/β-catenin signaling induced by mutations of genes, or aberrant activation of Wnt receptors trigger tumorigenesis in the colon, liver, skin, breast, bone marrow, and maybe more tissues , 86. It +T cells. β-Catenin expression in DCs negatively regulates antitumor immunity by inhibiting their cross-priming ability resulting in a dampened CD8+ T cell response [Wnt/β-catenin signaling also participates in the regulation of tumor immunology. By analyzing 266 metastatic human cutaneous melanoma samples, Spranger et al. revealed that tumor-intrinsic β-catenin activation dominantly excludes T cell infiltration into the melanoma tumor microenvironment. Therefore, intrinsic β-catenin activation may represent one mechanism of primary resistance to T cell-based immune-oncology therapies . Cross-presponse . Hence, Accumulating evidences suggest that Wnt/β-catenin signaling helps maintaining the stemness of cells in the embryo and the adult, as well as in cancer. The mammalian intestine contains a crypt compartment maintained by multipotent stem cells and a villus compartment extending into the lumen. Wnt signaling controls the proliferation of stem cells within the crypt base and thus drives the emergence of cells through the transit-amplifying zone into the villus. Indeed, Wnt target genes, such as LGR5, CD44 and Msi1, have become well-established markers of intestinal stem cells . In the The Wnt/β-catenin pathway has been proved to be crucial to various kinds of carcinoma. Therapeutic strategies targeting this signaling pathway are increasingly being proposed to aid the fight against cancer. Although no drugs specifically inhibiting this signaling pathway have been approved for the market, considerable efforts have been made towards their development. Inhibitors of the Wnt/β-catenin pathway can be classified into five categories according to the properties of the drugs: small molecules, peptides, antibodies, RNA interference and natural compounds. Figure In the Wnt/β-catenin signaling pathway, one target is the ligand—Wnt itself, which controls the activity of the pathway. Lipid modification of Wnt by membrane-bound o-acyltransferases-porcupine (PORCN) promotes its secretion and its binding ability with cognate receptors. LGK974, a potent and specific small-molecule inhibitor of PORCN demonstrates efficacy at well-tolerated doses in multiple tumor models, such as murine breast cancer and human head and neck squamous cell carcinoma in vivo. The phase I clinical study of LGK974 in patients with malignancies dependent on Wnt ligands is ongoing. In opposition to the role of Wnt-3a, Wnt-5a promotes the degradation of β-catenin. The formylated 6-amino-acid peptide fragment, (Foxy-5), mimics the effects of Wnt-5a and is currently being tested in a phase I clinical trial. Antibodies targeting the Wnt ligands and receptors are another method by which this signaling pathway is inhibited. OMP-18R5, OTSA-101 and OMP-54F28 are antibodies against multiple frizzleds, frizzled-10 and Wnt ligand respectively, and have all been enrolled in Phase I clinical trials .Axin, GSK3β and CK1 participate in the regulation of β-catenin degradation. Efforts are made to activate kinase activity and thus to reduce the stability of β-catenin. Axin is an important protein in promoting β-catenin degradation. Tankyrase1/2 is a kinase destabilizing Axin, which is chosen as a target to reduce the accumulation of β-catenin. G007-LK and JW55, inhibitors of TNK1/2, cause stabilization of Axin2 and thus increase degradation of β-catenin. The two small molecules are now in preclinical research. XAV939, another Axin stabilizer, has been in the phase I clinical trial to treat subjects with CRC, unfortunately it was stopped due to low enrollment –106. As Therapeutics targeting β-catenin cellular distribution is a promising potential strategy for the inhibition of Wnt/β-catenin pathway, although few inhibitors have yet been identified. One strategy would be to disturb the function of NPC, as β-catenin is transported through NPC during its subcellular localization either via a direct interaction or via an active, receptor-mediated mechanism. Wheat germ agglutinin (WGA), an inhibitor of NPC, has been reported to suppress nuclear transport of β-catenin . HoweverIn addition to NPC, nuclear import chaperones of β-catenin, such as Smad3/4, FOXM1, MUC1, IRS1, BCL-9 and LEF1, are also potential targets. Small molecules, especially peptides designed to interrupt the PPI of β-catenin and its specific import chaperones in specific tumor types, reduce the accumulation of β-catenin in the nucleus and suppress its transcriptional activity. For example, a peptide targeting BCL-9, an import chaperone as well as a transcriptional co-activator of β-catenin, has been developed  (discuss2) to bind with TCF4, Cadherin and APC. Moreover, the dissociation constant (Kd) of the β-catenin–TCF PPI is much lower than those of the β-catenin-cadherin and β-catenin–APC PPIs, making it difficult to obtain a highly selective inhibitor. β-Catenin function loss in cadherin junctions increases cell migration and induces metastasis. Furthermore, dissociation of APC from β-catenin results in its nuclear accumulation. These facts emphasize that obtaining high selectivity is a major concern for drug development. Recently, J. Leon Catrow et al have discovered a series of compounds with much higher selectivity for the β-catenin–TCF interaction over β-catenin–Cadherin and β-catenin–APC interactions. For example, PKF115-584 and CGP049090, which are obtained through optimization of the Acyl Hydrazone Moiety [High frequency of Wnt pathway gene mutations enhances the β-catenin transcriptional activity independent of upstream factors, which would limit the applicability of some therapeutic modalities mentioned above. Therefore, more efforts have been made to directly attenuate the function of β-catenin in the nucleus. As β-catenin functions as a transcriptional activator in a complex with TCF4, P300/CBP, BCL9 and so on, small molecules targeting these PPIs have a potentially bright future. More than thirty compounds and a stapled α-helical peptide have been reported to interfere with the β-catenin–TCF4 interaction –117. Thee Moiety , 119. Ote Moiety . Phase Ie Moiety . Takada e Moiety . Thus taBased on early discoveries linking the activation of Wnt/β-catenin signaling to HCC, breast and colon carcinomas, it has generally been accepted that aberrant activation of Wnt signaling promotes tumor initiation and progression. The activity of β-catenin is under the control of several highly regulated processes, including ligand–receptor interactions, β-catenin stability, β-catenin nuclear translocation and β-catenin transcriptional activity. Future studies aimed at determining the mechanisms that control these processes will be necessary to identify pivotal signaling nodes that could be targeted by therapeutic interventions.The complexity of the Wnt signaling pathway offers multiple levels of therapeutic intervention and currently there are a plethora of interventional approaches being explored to reduce Wnt/β-catenin signaling output. For example, great efforts have been made to interrupt upstream Wnt signaling events, including Wnt lipidation, Wnt secretion, and ligand-receptor interactions . Some co


SOX9, COL2A1, and ACAN, and matrix metalloproteinase genes MMP1/3/9/13, but increased ADAMTS 4/5. Inhibition of WNT signaling by PKF increased the expression of SOX9, COL2A1, ACAN, and MMP9, but decreased MMP13 and ADAMTS4/5. In addition, a high level of WNT signaling induced the expression of hypertrophic markers COL10A1, ALPL, and RUNX2, the dedifferentiation marker COL1A1, and glycolysis genes GULT1 and PGK1. Deposition of glycosaminoglycan (GAG) and collagen type II in the pellet matrix was significantly lost in the BIO-treated group and increased in the PKF-treated group. The protein level of COL10A1 was also highly induced in the BIO group. Interestingly, BIO decreased the number of apoptotic cells while PKF significantly induced apoptosis during chondrogenesis. The natural WNT antagonist DKK1 and the protein level of MMP1 in the pellet culture medium were decreased after PKF treatment. All of these chondrogenic effects appeared to be mediated through the canonical WNT signaling pathway, since the target gene Axin2 and other WNT members, such as TCF4 and β-catenin, were upregulated by BIO and downregulated by PKF, respectively, and BIO induced nuclear translocation of β-catenin while PKF inhibited β-catenin translocation into the nucleus. We concluded that addition of BIO to a chondrogenic medium of hMSCs resulted in a loss of cartilage formation, while PKF induced chondrogenic differentiation and cartilage matrix deposition and inhibited hypertrophic differentiation. However, BIO promoted cell survival by inhibiting apoptosis while PKF induced cell apoptosis. This result indicates that either an overexpression or overinhibition of WNT signaling to some extent causes harmful effects on chondrogenic differentiation. Cartilage tissue engineering could benefit from the adjustment of the critical level of WNT signaling during chondrogenesis of hMSC.Mesenchymal stem cells (MSCs) are multipotent cells, mainly from bone marrow, and an ideal source of cells in bone and cartilage tissue engineering. A study of the chondrogenic differentiation of MSCs is of particular interest for MSCs-based cartilage regeneration. In this study, we aimed to optimize the conditions for the chrondogenic differentiation of MSCs by regulating WNT signaling using the small molecule WNT inhibitor PKF118-310 and activator BIO. Human mesenchymal stem cells (hMSCs) were isolated from bone marrow aspirates and cultured in hMSCs proliferation medium. Pellet culture was subsequently established for three-dimensional chondrogenic differentiation of 5 weeks. WNT signaling was increased by the small molecule glycogen synthase kinase-3 inhibitor 6-bromoindirubin-3-oxim (BIO) and decreased by the WNT inhibitor PKF118-310 (PKF). The effects of BIO and PKF on the chondrogenesis of hMSCs was examined by real-time PCR, histological methods, and ELISA. We found that activation of canonical WNT-signaling by BIO significantly downregulated the expression of cartilage-specific genes  The treatment of injuries to hyaline cartilage is considered a challenge due to the limited ability of self-repair of articular cartilage. Although much research effort has been made to improve the regeneration of articular cartilage, there is no curative treatment available yet for rebuilding native articular cartilage. Human mesenchymal stem cells (hMSCs) are multipotent cells mainly from the bone marrow. They can be expanded in vitro and be differentiated into the osteogenic, chondrogenic, and adipogenic lineages , and areWNT signaling plays an extremely important role in the regulation of cell proliferation and differentiation in embryonic and cartilage development ,5. When Several small molecules that regulate WNT signaling have been reported . In our The potential effects of small molecules that activate/inhibit WNT/β-catenin signaling on cell culture have been demonstrated. Some studies have reported that BIO could promote cell growth ,17,18,19Even though many studies focus on small inhibitors or activators of WNT/β-catenin signaling in cell research, there is no report about the effect of an inhibitor/activator on hMSCs chondrogenesis in vitro. The aim of this study was to assess the potential effects of small molecules on chondrogenic-differentiating hMSCs with continuous inhibition/activation of canonical WNT signaling using a three-dimensional (3-D) pellet culture system.In order to test the efficacy and specificity of BIO and PKF in terms of activating/blocking WNT/β-catenin signaling, dose-response experiments were performed in HEK293t cells transfected with the TCF/LEF reporter. The cells were cultured in the medium with or without BIO/PKF. We found a dose-dependent increase in reporter activity when the cells were treated with BIO. Maximal activation was found at 2 µM. PKF treatment resulted in a 10-fold decrease in reporter activity at a concentration of 3 µM A. On theAXIN2, and the WNT-related genes TCF4 and CTNNB1 (gene encoding β-catenin protein), were measured by qPCR. The expression and nuclear translocation of β-catenin was detected by immunohistochemistry (IHC). We observed that BIO significantly induced mRNA expression of the canonical WNT target gene AXIN2 and the WNT signal related genes TCF4 and CTNNB1, while PKF effectively inhibited canonical WNT signaling  assay A–C indicIt is evident from the qPCR result that BIO significantly induced the expression of hypertrophic markers. We assumed that the treatment of BIO might induce cells towards hypertrophic differentiation. In order to investigate hypertrophic differentiation, the typical hypertrophy marker type X collagen was detected by immunohistochemistry. Type X collagen could hardly be detected in either the control or the PKF group. However, BIO greatly induced the expression of COL10A1 A.During the process of hypertrophic differentiation, cells will enlarge, terminally differentiate, and ultimately undergo mineralization. In order to further elucidate this process, Alizarin red S staining was performed to evaluate mineralization. No matrix mineralization was observed in any of the groups B.Several members of the WNT family, such as WNT16, WNT7a, and TCF4, have been shown to be involved in the regulation of chondrocyte apoptosis ,27,28. IPreviously, we found that DKK1, as a natural WNT antagonist, plays a vital role in the regulation of chondrogenic differentiation of hMSCs. In this study, we collected the medium and measured the concentration of endogenous DKK1 secreted from hMSC pellets after treatment with BIO or PKF by ELISA. After continuous stimulation for 5 weeks, BIO slightly increased the expression of DKK1. However, PKF dramatically decreased DKK1 expression to a very low level A. The reCell-based cartilage regeneration often involves the use of chondrocytes or hMSCs. However, hMSCs chondrogenic differentiation is still unsatisfactory. An improvement in current cartilage tissue-engineering protocols requires more detailed insight into the molecular mechanisms that regulate the distinct steps of chondrogenic differentiation of hMSCs. We propose that targeting the canonical WNT signaling pathway to increase chondrogenic differentiation of MSCs would be a very important and a valuable strategy.In the present study, we have attempted to investigate the effect of two small molecules, BIO and PKF, which have been described as positive and negative regulators of the WNT signaling pathway ,13,14,15We found that the addition of the canonical WNT activator BIO downregulated the expression of cartilage-specific genes and increased cartilage matrix degradation. In contrast, addition of the WNT inhibitor PKF upregulated the expression of cartilage markers and increased the cartilage matrix deposition. This is consistent with other findings showing that the expression pattern of three cartilage-specific genes are downregulated in cultures with 1 µM BIO treatment in mice MSCs cells, while PKF increased the cartilage markers in mouse fetal metatarsals ,22.The effects of BIO and PKF on chondrogenic differentiation are mediated through the canonical WNT signaling pathway, as both factors modulate the WNT pathway via β-catenin. BIO functions through preventing β-catenin breakdown through the inhibition of GSK3β, while PKF prevents the interaction of β-catenin with transcription factors, TCF/LEF. The observed effects of BIO and PKF are in line with the observations from transgenic animals in which β-Catenin is modulated ,30. We tMesenchymal stem cells show promising results in cell-based treatment for cartilage regeneration. Unfortunately, more and more studies are suggesting that the loss of the chondrogenic phenotype and expression of hypertrophic markers, including COL10A1, MMP13, and VEGF, are observed in chondrogenically differentiated hMSCs ,32,33. TMMP 1/3/13 in human MSCs [MMP1/3/9/13 in hMSCs. The WNT inhibitor PKF was found to decrease MMP13, but increased the MMP9 expression. Although MMP13 is one of the proteinases, it is also a typical hypertrophic marker. Its expression can be decreased by alternative pathways, such as the RUNX2- and FGF2-mediated pathways [MMPs are the main proteinases degrading collagens and proteoglycans in joint disease. Previous research by our group shows that, in human chondrocytes, WNT/β-catenin is a potent inhibitor of MMP expression in both basic and IL1β-treated conditions. The results also show that WNT3a decreased the mRNA expression of man MSCs ,39. Simipathways . The MMPCOL1A1 expression, which further proves the role of WNT/β-catenin signaling in chondrocyte dedifferentiation. In addition, considering the metabolic ATP requirement of MSCs shifts from oxidative phosphorylation to predominantly glycolytic metabolism during chondogenic differentiation [GLUT1 and PGK1, were detected in this study. Interestingly, their expression was elevated in BIO group but dramatically decreased in the PKF group, which may indicate that the glucolysis metabolism in PKF-treated cells was inhibited greatly.It has been shown that overexpression of β-catenin, using a β-catenin activator or in APC knockout mice, leads to the dedifferentiation of chondrocytes ,42. We fntiation , two keyMineralization is the ultimate end stage of chondrocyte hypertrophy. To investigate whether BIO-treated chondrogenically differentiating MSCs also undergo matrix mineralization, Alizarin red S staining was adopted to observe the mineralization nodule. No mineralization was observed in either the control or the experimental group in this study, which suggests that the hypertrophic differentiation did not reach the end stage. In addition, it has been shown that chondrocyte hypertrophy does not have to be related to chondrocyte mineralization .Apoptosis refers to the autonomous and orderly death of gene-controlled cells in order to maintain a stable internal environment, and is an active process involving the activation, expression, and regulation of a range of genes without inducing inflammation . One stuActivated WNTβ-catenin signaling is well-known to stimulate cell proliferation in multiple cell cultures in vitro ,18,51,52SOX9, COL2A1, and ACAN, was inhibited by BIO-induced high WNT signaling, while being promoted by the lower WNT activity after PKF treatment. Moreover, active canonical WNT signaling induced the expression of chondrocyte hypertrophic markers, such as RUNX2, COL10A1, and ALPL, while their expression was reduced by lowering the WNT activity  together with lentiviruses constitutively expressing Renilla luciferase (SABiosciences). HEK293t cells were seeded at 8000 cells/cm2 in 96-wells plates  and cultured for 24 h in basic medium (DMEM supplemented with 10% FBS and 100 µ/mL penicillin and streptomycin). The cells were treated with BIO  or PKF118-310 (PKF) (Sigma Aldrich) for 24 h. After 24 h, the cells were lysed in Glo lysis buffer and luciferase activity was measured using a Dual-Glo luciferase assay kit .HEK293t cells were infected with Cignal2. MSCs were selected by adherence in a proliferation medium .The use of human bone marrow aspirates was approved by a local medical ethics committee  and written informed consent by the donors was obtained. The aspirates were resuspended using a 20 Gauge needle and plated at a density of 0.5 million mononucleated cells/cm−7 M dexamethasone, 100 U penicillin/mL, and 100 μg/mL streptomycin) and centrifuged for 5 min at 500× g. Three groups were included in this experiment: hMSC pellets in chondrogenic differentiating medium as the control group, hMSC pellets with BIO treatment, and hMSC pellets with PKF treatment. BIO and PKF were used at a concentration of 2 and 3 µM, respectively. Three donors were used for each condition as biological triplicates. Cell pellets were grown at 37 °C in a humid atmosphere with 5% CO2. The medium was refreshed twice a week. The cell pellets were cultured for 5 weeks before analysis.To obtain pellet-cultures, 250,000 MSCs at passage three were seeded in 10 mL round-bottom tubes in chondrogenic differentiation medium  according to the manufacturer’s protocol. The concentration and purity of the RNA samples were measured with the Nanodrop2000. The total RNA was reverse-transcribed into cDNA using the iScript cDNA Synthesis kit . Quantitative PCR (qPCR) was performed as .Chondrogenically differentiated hMSC pellets were fixed with 10% buffered formalin for 15 minutes (min) and embedded in paraffin using routine procedures. Sections of 5 μm were cut using a microtome . Alcian blue/Safranin O staining was performed as described previously . The sec®Fluor 546-labelled goat anti-rabbit antibody in 5% BSA in PBS was added and incubated for 2 h at RT. The samples were rinsed with PBS, and mounting medium with DAPI was added. The slides were viewed by BD pathway™ confocal microscopy .For the immunofluorescent staining of collagen type II, 5 µM sections were de-paraffinized in xylene and rehydrated with graded ethanols. The samples were pre-incubated with 5 μg/mL proteinase K (Sigma Aldrich) for 10 min at room temperature (RT) followed by 1 mg/mL hyaluronidase (Sigma Aldrich) for 40 min at 37 °C. They were blocked in 5% BSA in PBS for 1 h, after which they were incubated overnight at 4° with rabbit anti-human collagen II antibody , which was diluted 1:100 in 5% BSA in PBS. The cells were rinsed three times with PBST, 5 min/time. Then, AlexaThe immunohistochemistry staining of β-catenin and Collagen type X was performed using 5 μm sections. The sections were pre-incubated with 5 μg/mL proteinase K (Sigma Aldrich) for 10 min followed by 1 mg/mL hyaluronidase (Sigma Aldrich) for 40 min at 37 °C. Rabbit anti-human β-catenin antibody  or mouse anti-human Collagen type X antibody  was diluted 1:100 in PBS and incubated overnight at 4 °C. Non-immune controls underwent the same procedure without primary antibody incubation. The biotinylated secondary antibody was diluted 1:500 in 5% BSA in PBS and incubated for 30 min at RT. horseradish peroxidase (HRP)-Streptavidin was added and incubated for 30 min at RT. A DAB substrate kit  was used for visualization. The cell nucleus was counterstained with hematoxylin for Collage type X detection. Images were taken by using a Nanozoomer .TM fluorometric TUNEL System (Promega). Nuclei were counterstained with Hoechst 33342. Images were viewed by means of BD pathway confocal microscopy.Apoptosis of chondrocytes was detected using The DeadEnd® dsDNA System kit (Promega) according to the manufacturer’s instructions.After 5 weeks of chondrogenic differentiation, the pellets were predigested in 250 µL of Tris-HCl buffer  with 1 mg/mL proteinase K (Roche) for 16 h at 56 °C. Diluted samples (25 μL) were mixed with 150 μL 1,9-dimethylmethylene blue (DMMB) dye solution  and absorbance was measured at 525 nm. The relative cell number was determined by quantification of the total DNA using a QuantiFluor2SO4 and measured at 450 nm of the microplate reader.The culture medium for pellet samples was collected weekly. The protein concentration of DKK1 was measured using ELISA according to the manufacturer’s guidelines . Secreted MMP1 in medium was also measured by ELISA using mouse anti-human MMP1 antibody , which was followed by incubation with a rabbit anti-mouse antibody coupled to a peroxidase. The amount of HRP was developed by the addition of Tetramethylbenzidine . The reactions were stopped by the addition of Ht-tests or one-way ANOVA. p < 0.05 was considered statistically significant.For the experiments using human bone marrow, samples were obtained from three donors. Each experiment was performed in triplicate. Data is expressed as the mean ± SD and analyzed by two-tailed student’s


Enterococcus faecalis-infected macrophages increased Wnt3α expression while suppressing Wnt inhibitor factor 1 (Wif1). Wnt/β-catenin activation was confirmed by increased active β-catenin and Tcf4. in vivo, active β-catenin was evident in colon biopsies from E. faecalis-colonized Il10 knockout mice compared to sham-colonized mice. This effect was mediated, in part, by 4-hydroxy-2-nonenal and tumor necrosis factor α. MIBE also activated pluripotent transcription factors c-Myc, Klf4, Oct4, and Sox2 in YAMC cells and colons from E. faecalis-colonized Il10 knockout mice. These transcription factors are associated with cellular reprogramming, dedifferentiation, and induction of colorectal CSC progenitors. In support of this was an increase in the expression of Dclk1 and CD44, two colorectal CSC markers, in YAMC cells that were exposed to MIBE. Finally, compared to normal colon biopsies and hyperplastic polyps, DCLK1 expression increased in human tubular adenomas and invasive colorectal cancers. Blocking β-catenin/TCF4 signaling using FH535 and CTNNB1-specific small interfering RNA decreased DCLK1 expression in HCT116 human colon cancer cells. These findings provide mechanism for microbiome-induced colorectal cancer and identify new potential targets for colorectal cancer prevention.The colonic microbiome contributes to the initiation of colorectal cancer through poorly characterized mechanisms. We have shown that commensal-polarized macrophages induce gene mutation, chromosomal instability, and endogenous transformation through microbiome-induced bystander effects (MIBE). In this study we show that MIBE activates Wnt/β-catenin signaling and pluripotent transcription factors associated with dedifferentiation, reprogramming, and the development of colorectal cancer stem cells (CSCs). Exposure of murine primary colon epithelial cells (YAMC) to  Cancer stem cells (CSCs) play pivotal roles in major solid tumor heterogeneity, tumor growth and metastasis, and resistance to chemotherapy . The celApc) in intestinal stem cells are associated with cellular transformation and tumor formation. This suggests that these cells, or long-lived descendants, are cells of origin for CSCs [Apc mutations along with dysregulated Wnt/β-catenin signaling are drivers for the development of CSCs [Bacteroides fragilis and Fusobacterium nucleatum can activate Wnt/β-catenin signaling [In murine models of colorectal cancer deletions and/or mutations of the adenomatous polyposis coli gene , a DNA mutagen and mitotic spindle inhibitor derived from the peroxidation of ω6 polyunsaturated fatty acids . Tumor nin vitro studies confirmed 4-HNE and TNFα as independent drivers of these markers. Finally, we noted increased DCLK1 expression in association with Wnt/β-catenin signaling in human tubular adenomas and invasive colorectal cancers, but not normal colon tissue. These findings demonstrate that MIBE activates Wnt/β-catenin signaling and induces pluripotent transcription factors associated with dedifferentiation, reprogramming, and transformation of primary colon epithelial cells.In this study we found that MIBE activated Wnt/β-catenin signaling and multiple pluripotent transcription factors. These transcription factors were associated with the expression of CSC markers. E. faecalis-infected macrophages that were polarized to an M1 state by E. faecalis [Ctnnb1 expression was seen 48 hrs following exposure , and inactive p-Gsk3β (Ser9) 24 to 72 hrs following co-culture  were co-cultured with uninfected or faecalis , 17. As exposure . In conte Figure . Notablys Figure , supports Figure . Of noteMC cells  and 3. Ts Figure . In contE. faecalis-colonized Il10-/- mice—a model for MIBE leading to colorectal cancer—developed colitis and colorectal cancer after 9 months of colonization [E. faecalis at ~109 colony-forming units per gram feces during entire experiment [in vivo.nization . These mperiment . When coCtnnb1 at 5 to 48 hrs following 4-HNE treatment . Similarly, FH535 reduced TCF4 expression by 68% and 83% at 24 and 48 hrs post-treatment, respectively (P < 0.001). This resulted in 42% and 44% decreased DCLK1 expression at 24 and 48 hrs, respectively . Partial silencing of CTNNB1 resulted in a 61% decrease in DCLK1 expression for cells transfected with CTNNB1 siRNA compared to non-targeting siRNA . These results indicate that MIBE-induced Wnt/β-catenin signaling induces DCLK1 expression.To assess the role of β-catenin signaling in DCLK1 expression during colorectal carcinogenesis, we blocked this pathway in HCT116 cells using FH535, an inhibitor of β-catenin/Tcf4. Western blots showed 43% and 71% decreased β-catenin in treated cells at 24 and 48 hrs, respectively, compared to untreated controls . We found that 4-HNE and TNFα, two mediators of MIBE, each activated Wnt/β-catenin. This likely occurred by inducing Wnt3α and suppressing WIF1 suggesting post-translational activation of β-catenin. In addition, oxidative stress created by 4-HNE could also induce COX-2 in target cells. This enzyme produces 4-HNE as a byproduct of catalysis and the result would be a feedforward mechanism for further production and β-catenin activation [Wnt/β-catenin signaling occurs during normal stem cell development and tissue homeostasis , and alstivation . Similarright). These findings were consistent with Wnt activation driving CSC development [Sfrp2 expression in primary colon epithelial cells transformed by MIBE or purified 4-HNE [Nuclear β-catenin binds TCF4 and induces pluripotent transcription factors and CSC markers Figure , right. elopment , 29. Of elopment . Sfrp2 ielopment . We preved 4-HNE , 24. Alt9) to activate β-catenin in a fashion similar to that for YAMC cells [4-HNE and TNFα contribute to the initiation of colorectal cancer as diffusible mediators of MIBE , 20. In MC cells . BecauseMC cells , it remaMC cells . TNFα caMC cells . InteresMC cells , 38. In in vitro, synergistic effects could be anticipated since TNFα induces COX-2 and this enzyme is a source for 4-HNE [Inflammatory signaling through NF-κB helps stabilize β-catenin and facilitates bidirectional conversion of intestinal epithelial cells into stem-cell-like physiology through dedifferentiation . This isor 4-HNE . Indeed,or 4-HNE .Seminal work by Takahashi and Nakayama showed that somatic cells can be reprogrammed to pluripotent stem cells by only four transcription factors: c-Myc, Klf4, Oct3/4, and SOX2 . Each ofE. faecalis-infected macrophages or 4-HNE [Teratoma formation is the gold standard for induced pluripotency . In prioor 4-HNE , anaplase.g., Oct-4, Nanog, and Sox2 [in vitro and in vivo data showed that c-Myc, Klf4, Oct4, and SOX2 were each induced by MIBE. Although Nanog is a marker for pluripotent stem cells, we did not assess this transcription factor since is not required for somatic pluripotency in mice [An alternate approach to assess induced pluripotency involves measuring specific factors involved in dedifferentiation, and Sox2 . Both in in mice . In our  in mice , 24. Los in mice . Finally in mice , 26, 51.Two distinct models describe the morphogenesis of colorectal cancer: a “bottom-up” theory for CSCs arising from normal stem cells at the base of crypts , and a “S-transferases (GSTs) metabolize a wide range of electrophilic carcinogens [Il10-/- mice colonized with E. faecalis for only 2 weeks [Little is known about mechanisms that may potentially defend against MIBE. Glutathione cinogens . 4-HNE, cinogens . Inducti 2 weeks . Other pE. faecalis as a model microorganism for MIBE because E. faecalis produces extracellular superoxide [Il10-/- mice but contributes to colorectal cancer development [E. coli-infected macrophages also generate 4-HNE [Finally, we chose peroxide . This oxelopment . Nonethete 4-HNE  and, as In summary, MIBE activates Wnt/β-catenin signaling in primary colon epithelial cells. Associated with this finding was an enhanced expression of transcription factors involved in induced pluripotency and colorectal CSC markers. These results provide evidence that selected members of the intestinal microbiome can drive colorectal carcinogenesis through dedifferentiation and reprogramming and should help expand strategies for targeting CSCs.E. faecalis OG1RF and Escherichia coli DH5α were grown overnight in brain-heart infusion  and Luria-Bertani broth (BD), respectively, at 37°C and washed with phosphate buffered saline (PBS) prior to infecting macrophages [Murine primary colon epithelial cells , murine macrophages , and human colon cancer cells  were grown as previously described . E. faecrophages . FH535 wE. faecalis and E. coli was performed as previously described [E. faecalis OG1RF and E. coli DH5α were centrifuged, washed with sterile PBS, and colony-forming units determined. Macrophages were treated with E. faecalis OG1RF and E. coli DH5α at a multiplicity of infection (MOI) of 1,000 and 100, respectively, in antibiotic-free medium for 1 hr at 37°C. As previously described, cells were washed with sterile PBS and numbers counted prior to use in a Transwell dual-chamber co-culture system  with macrophages growing on an insert and separated from YAMC cells by a membrane with 0.4 μM pores [Infection of macrophages with escribed . RAW264.μM pores . YAMC ce5) were grown overnight in 6-well plates and treated with purified 1 μM 4-HNE for 1 hr or 100 ng/ml murine TNFα for the duration of experiments at 33°C. Cells were washed and RNA or protein extracted at each time point.For treatments with 4-HNE or TNFα, YAMC cells  were colonized with E. faecalis OG1RFSS (n = 7) or PBS sham (n = 7) as previously described [Animal studies were approved by the University of Oklahoma Health Sciences Center and Oklahoma City VA Health Care System animal care and use committees. Specific pathogen-free escribed . Mice weCTNNB1/Ctnnb1, DCLK1/Dclk1, and ACTB/Actb  per manufacturer's instruction. qRT-PCR was carried out on the CFX96 Real-Time System  using SYBR Premix Ex Taq II  at 95°C for 30 sec followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec. Expression of CTNNB1/Ctnnb1 and DCLK1/Dclk1 were normalized to ACTB/Actb and relative gene expression calculated by control.RNA isolation and qRT-PCR were performed as previously described . PrimersCTB/Actb  were synE. faecalis-infected macrophages in dual-chamber co-culture system for 48 hrs at 33°C. Cytoplasmic and nuclear protein was extracted from YAMC cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents  according to the manufacturer's instructions.YAMC cells were exposed to untreated macrophages or TM Western ECL Substrate (Bio-Rad) and captured by ChemiDoc XRS+ system (Bio-Rad).Whole-cell extracts (20 μg) were separated by SDS-PAGE and blotting performed as previously described . The souEpitope retrieval for IHC and IF staining were performed as previously described . PrimaryCTNNB1-specific siRNAs were purchased from Cell Signaling Technology and scrambled nontargeting siRNA from GE Dharmacon . Transient transfections were conducted using lipofectamine® 3000 reagent and gene silencing confirmed by qRT-PCR and Western blotting.Silencing of β-catenin was performed using small interfering RNA (siRNA) as previously described . Human Ct test was used for comparisons between experimental and control groups. P values < 0.05 were considered significant.Data were expressed as means with standard deviations (SD) shown for at least 3 independent experiments. Student's


Vertically aligned p-type silicon nanowire (SiNW) arrays were fabricated through metal-assisted chemical etching (MACE) of Si wafers. An indium tin oxide/indium zinc oxide/silicon nanowire (ITO/IZO/SiNW) heterojunction diode was formed by depositing ITO and IZO thin films on the vertically aligned SiNW arrays. The structural and electrical properties of the resulting ITO/IZO/SiNW heterojunction diode were characterized by field emission scanning electron microscopy (FE-SEM), X-ray diffraction (XRD), and current−voltage (I−V) measurements. Nonlinear and rectifying I−V properties confirmed that a heterojunction diode was successfully formed in the ITO/IZO/SiNW structure. The diode had a well-defined rectifying behavior, with a rectification ratio of 550.7 at 3 V and a turn-on voltage of 2.53 V under dark conditions. Nanostructured solar cells  is an n-type semiconductor with a large binding energy of 60 meV and a wide bandgap of 3.3 eV in the UV range. ZnO has numerous applications in optoelectronic devices, including ultraviolet (UV) visible photodetectors ,14, solaIn this work, we fabricated silicon nanowires using a top-down method: metal-assisted chemical etching (MACE) . This ap2; the slices were subsequently cleaned ultrasonically in acetone, isopropyl alcohol, and deionized water, then dried with nitrogen (N2) gas, as shown in 3) (HF:AgNO3 = 5:0.02 M) to deposit silver (Ag) particles, which acted as the catalyst in the following etching process. Subsequently,  aqueous solution for 90 s. Finally, the SiNWs were rinsed with deionized water and blown dry in N2. 2O2 = 98:2 mol % ceramic target  using a radio frequency (RF) magnetron sputtering system. The working distance between the SiNW substrate, and the target was fixed at 15 cm. The base pressure was 8 × 10−6 torr, and the working pressure was 2 × 10−2 torr. The deposition temperature of the IZO thin films was room temperature, the RF power was 100 W, and the deposition time was 1 h. The ITO thin films were then deposited on the IZO/SiNW substrates under the same deposition conditions, except that the deposition time was extended to 2 h (P-type silicon nanowires (SiNWs) were fabricated through metal-assisted chemical etching (MACE) . Figure quently, b,d, thisd to 2 h f. Finalld to 2 h g. The moThe surface morphologies of the ITO, IZO, and ITO/IZO thin films deposited on the Si substrate are shown in 2O3 phase was found. As shown in The X-ray diffraction (XRD) patterns of the ITO and IZO films deposited on the Si substrate are presented in The SEM images in Current–voltage (I–V) characterization of the IZO/Si and ITO/IZO/Si was carried out at room temperature, as shown in −9 A/cm2 at 1 V for the ITO/IZO/Si heterojunction diode and smaller than the IZO/Si heterojunction diode. The roles of the ITO layer are interpreted with the help of the following key parameters: The electrode area ratio of the ITO/IZO/Si heterojunction diode is much larger than that of the IZO/Si heterojunction diode. As the ITO layer covers the entire surface of the IZO/Si, although the resistivity of ITO film (5 × 10−3 Ω·cm) is larger than that of Al film (10−5 Ω·cm), the generated carriers are readily collected via the shortest path between the p–n heterojunction and the ITO layer and effectively transported to the Al finger electrode via ITO without recombination. Therefore, the ITO/IZO/Si heterojunction diode obtained the rectifying I–V curve and small leakage current.Cmax) of the ITO/IZO/SiNW heterojunction diode is about 6.21 nF/cm2 and is larger than that of the ITO/IZO/Si heterojunction diode (about 3.58 nF/cm2). The ratio of Cmax is proportional to the ratio of the p–n junction areas of the ITO/IZO/SiNWs and the ITO/IZO/Si heterojunction diode [To improve the properties of the heterojunction diode, SiNWs were used. on diode . It is uIs is the saturation current; k is the Boltzmann constant; n is the ideality factor; T is the temperature in Kelvin; q is the electron charge; and V is the applied voltage. The Is of the heterojunction diode is expressed by the following equation [A is the device area; A* is the Richardson constant; and The nonlinear I–V characteristics with diode behavior can be described by a thermionic emission (TE) model theory. The current in such a device can be expressed as (2)I=Is[equation :(3)Is=An of the ITO/IZO/SiNW heterojunction diode were calculated. The n = 1.53 can be calculated from the slope of the linear region of the I–V curve in the forward bias region, and the value of Using Equations (2)–(4), the values of al layer .n = 1.53) can be calculated from the slope of the linear region of the I–V curve in the forward bias region and the barrier height  characteristics confirmed that a p–n junction structure had been formed in ITO/IZO/Si and ITO/IZO/SiNW heterojunction structures. To enhance the properties of the ITO/IZO/Si heterojunction diodes, an array of vertically aligned SiNWs was fabricated through metal-assisted chemical etching. Subsequently, ITO and IZO thin films were de


In the bi-articular no-spring condition the pneumatic muscles were also attached to the thigh segment but with a non-elastic cord. In the mono-articular condition the pneumatic muscles were attached to the shank segment. We found the highest reduction in metabolic cost of 13% compared to walking with the exoskeleton powered-off in the bi-articular plus spring condition. Possible explanations for this could be that the exoskeleton delivered the highest total positive work in this condition at the ankle and the knee and provided more assistance during the isometric phase of the biological plantarflexors. As expected we found that the bi-articular conditions reduced m. gastrocnemius EMG more than the mono-articular condition but this difference was not significant. We did not find that the mono-articular condition reduces the m. soleus EMG more than the bi-articular conditions. Knowledge of specific effects of different exoskeleton configurations on metabolic cost and muscle activation could be useful for providing customized assistance for specific gait impairments.The bi-articular m. gastrocnemius and the mono-articular m. soleus have different and complementary functions during walking. Several groups are starting to use these biological functions as inspiration to design prostheses with bi-articular actuation components to replace the function of the m. gastrocnemius. Simulation studies indicate that a bi-articular configuration and spring that mimic the m. gastrocnemius could be beneficial for orthoses or exoskeletons. Our aim was to test the effect of a bi-articular and spring configuration that mimics the m. gastrocnemius and compare this to a no-spring and mono-articular configuration. We tested nine participants during walking with knee-ankle-foot exoskeletons with dorsally mounted pneumatic muscle actuators. In the  In most mainstream human-like robots  ankle-foot orthoses do not sufficiently replicate the function of the m. gastrocnemius  and a bi-articular spring  during walking at 1.25 msl Figure . The walThe participants wore bilateral hinged knee-ankle-foot exoskeletons powered by pneumatic artificial muscles Figure , Movie 1The height of the hinge joint for the knee joint was adjusted to match the participants' anthropometry. The exoskeleton was attached to the wearer by means of Velcro straps and tape around the thigh and lower leg segment. The lower leg segment of the exoskeleton weighed 0.65 kg per side, and the thigh segment of the exoskeleton weighed 1.25 kg per side. The design of our new bi-articular exoskeleton was based on our previous ankle exoskeleton  was used to trigger the onset and end of the pneumatic muscle contraction at different percentages of the stride cycle based on signals from heel switches . The pneumatic muscles were made to contract and lengthen by opening and closing of pneumatic valves . A constant supply pressure of 3.5 bar was used. The exact behavior of the pneumatic muscles was dependent on the inflation and deflation of the pneumatic muscles, the force-pressure-length relationship and the kinematics and the kinetics of the participant. This actuation control system was similar to the system used in Malcolm et al. .We tested five conditions Figure , Movie 1bi-articular plus spring condition, we attached the proximal end of the pneumatic muscles to the medial and lateral sides of the thigh segment via an elastic cord and a second non-elastic cord in parallel with the elastic cord that served to limit the maximum elongation. When pilot testing the bi-articular plus spring condition, we determined based on subjective perception that this second non-elastic rope was necessary to achieve an assistive effect during push-off because pneumatic muscles can only contract up to about 30% of their resting length. We intended this configuration to assist both during the eccentric and the concentric phase of the m. gastrocnemius contraction.In the bi-articular no-spring condition, we attached the proximal end of the pneumatic muscles to the medial and lateral side of the thigh segment only via a non-elastic elastic cord. We intended this configuration to assist mostly with the concentric phase of the m. gastrocnemius contraction.In the mono-articular no-spring condition, we attached the proximal end of the pneumatic muscles to the shank. This attachment configuration was roughly similar to that used in our previous study  we positioned the pneumatic muscles such that they had a moment arm of ~11 cm vs. the ankle joint. In the bi-articular conditions, the proximal attachment was positioned on the thigh such that in the standing position, the moment arm vs. the knee joint was about half of the moment arm vs. the ankle in order to mimic the biological moment arm ratio of the m. gastrocnemius  and the bi-articular no-spring and mono-articular condition would reach maximum elongation at the beginning of the concentric phase of the plantarflexors.In all ius Hof, . In all active condition. In one trial, we gradually shifted the onset timing from 23 to 53% of the stride until the participant verbally indicated that the actuation onset timing went past their perceived optimum. In another trial, we followed the same procedure in the opposite direction. We then used the mean values from the ascending and descending trials as control inputs for further metabolic and biomechanical testing. For the actuation ending, we always used a fixed control input of 60% of the stride. In the metabolic and biomechanical testing protocols, participants walked 4 min in each condition and rested while we changed the configurations. We randomized the order of the conditions.Before the metabolic and biomechanical testing protocol, the participants were allowed 18 min . We recorded the force from the pneumatic muscles at a rate of 1,000 fps with a load cell  mounted on the distal end of the pneumatic artificial muscles such that the load cell registered the total force from the pneumatic muscle (and the series elastic cord and/or non-elastic cord in the bi-articular conditions). The load cell data from one participant are missing due to a device malfunction. We recorded the muscle activation on the right leg of the m. tibialis anterior, soleus, gastrocnemius medialis, gastrocnemius lateralis, vastus lateralis, rectus femoris, biceps femoris, and gluteus maximus at a rate of 1,000 fps using surface EMG sensors . We measured the kinematics of the right leg using sagittal video recording at a rate of 200 fps  and reflective markers on the forefoot, ankle, knee and trochanter. We recorded heel contact times using the foot switches of the exoskeleton. We processed indirect calorimetry measurements for the last 2 min of each 4-min condition. We collected load cell, EMG, kinematic, and temporal data only during the exoskeleton conditions  over a 10-second period during the last minute of each condition.We recorded respiratory Opowered-off condition. We filtered the marker data with a 12-Hz Butterworth lowpass filter. Based on visual inspection, we excluded the EMG data from 24 trials  due to the presence of artifacts. We calculated the sagittal plane joint angles and angular velocities of the ankle, knee, and hip joints. For each joint angle, we subtracted the joint angle in the standing position. We estimated the total exoskeleton ankle moment by multiplying the load cell force by the moment arm vs. the ankle. In the bi-articular condition, we estimated the exoskeleton knee moment by multiplying the load cell force by the moment arm vs. the knee. We calculated the total exoskeleton power vs. the ankle and the knee by multiplying the exoskeleton moments by the joint angular velocities, and we calculated the positive exoskeleton work rates by integrating the positive portions of the exoskeleton power over time and dividing by the stride time. We calculated the step length by multiplying the step times obtained from foot switches by the speed of the treadmill. All time-series data were normalized vs. the stride time based on heel contact detection by the foot switches. We calculated the minima and maxima from all normalized stride time data, and we calculated the onset timing from the exoskeleton ankle moment data.We calculated the metabolic rate using the Brockway equation  and the actual metabolic cost. All statistical tests were conducted in MATLAB .For each time series and metric, we calculated the mean and standard error. For each metric, we tested whether there were any effects of exoskeleton condition using repeated measures ANOVA. We used Mauchly's test to verify sphericity and used the Greenhouse-Geiser correction if the sphericity assumption was violated. If the repeated measures ANOVA indicated a significant effect, we evaluated pairwise differences using paired bi-articular plus spring condition, bi-articular no-spring condition, and mono-articular no-spring condition, respectively . The selected actuation onset was significantly earlier in the bi-articular plus spring condition than in the mono-articular no-spring condition . The selected actuation onset in the mono-articular no-spring condition was not significantly different from the average value from previous publications in which similar mono-articular soleus-mimicking configurations were used .Participants selected actuation onsets of 36.1 ± 1.6, 38.4 ± 1.3, and 40.9 ± 0.9% in the −1 in the bi-articular plus spring condition, bi-articular no-spring condition, and mono-articular no-spring condition, respectively. Exoskeleton configuration had a significant effect on the peak exoskeleton moment . The peak exoskeleton ankle moment in the bi-articular plus spring condition was higher than that in the bi-articular no-spring condition . The elastic cord in the bi-articular plus spring condition resulted in an average angular stiffness of 2.662 Nm°−1 around the ankle during the phase before the pneumatic muscle contraction.The peak exoskeleton ankle moments were 0.35 ± 0.04, 0.26 ± 0.04, and 0.33 ± 0.03 Nm kg−1 per side in the bi-articular plus spring condition, bi-articular no-spring condition, and mono-articular no-spring condition, respectively. The peak exoskeleton positive knee work rates were 0.110 ± 0.011 and 0.070 ± 0.013 W kg−1 per side in the bi-articular plus spring condition and bi-articular no-spring condition, respectively.The peak exoskeleton positive ankle work rates were 0.065 ± 0.006, 0.044 ± 0.006, 0.103 ± 0.011 W kg−1 in the bi-articular plus spring condition, 3.04 ± 0.15 W kg−1 in the bi-articular no-spring condition, 3.02 ± 0.12 W kg−1 in the mono-articular no-spring condition, 3.21 ± 0.13 W kg−1 in the powered-off condition, and 2.77 ± 0.12 W kg−1 in the no-exoskeleton condition  on the net metabolic rate. The metabolic rate in the bi-articular plus spring condition was 12.8 ± 3.1% lower than that in the powered-off condition , and lower than that in the bi-articular no-spring condition . The metabolic rate in the bi-articular no-spring condition was on average 5.6 ± 2.7% lower than in the powered-off condition but this difference was not significant . The metabolic rate in the mono-articular no-spring condition was 5.7 ± 2.5% lower than in the powered-off condition . The metabolic rate was 13 ± 4% lower in the no-exoskeleton condition than in the powered-off condition . None of the exoskeleton conditions reduced the metabolic rate below that of the no-exoskeleton condition.Net metabolic rates were 2.79 ± 0.12 W kgdf = 30, T = 0.212, p = 0.042, R2 = 0.126, mixed-model ANOVA), positive knee work , and positive total joint work   and biceps femoris   and mono-articular no-spring condition  were lower than in the powered-off condition.The peak m. soleus EMG-values in the bi-articular no-spring condition was also lower than that in the powered-off condition  and that in the bi-articular plus spring condition .The peak m. biceps femoris in the dfcond. = 3, dferr. = 24, F = 44.481, p < 0.001, η2 = 0.848), maximum dorsiflexion , and maximum knee extension  . Maximum plantarflexion in the bi-articular plus spring condition was higher than that in the powered-off condition . In all active exoskeleton conditions, the maximum dorsiflexion angle before push-off was lower than that in the powered-off condition .Maximum plantarflexion in the mono-articular no-spring condition was higher than those in all the other exoskeleton conditions . We found no significant effects of exoskeleton configuration on hip joint angle or step length.Maximum knee extension (just before push-off) in the bi-articular conditions the coupling between the knee and ankle angle was more similar to the powered-off condition than in the mono-articular condition . Calculating the power from each component separately would allow to discuss how each component contributes to the power delivered by the exoskeleton (Eslamy et al., A limitation of our study is that the timing for each condition was selected based on perception tests. The perceived optimal timing in the Another reason why we did not find reductions in metabolic cost below that of walking without an exoskeleton may have been the additional mass of the thigh segments of the exoskeleton. While our exoskeleton weighed relatively less than other knee-ankle-foot exoskeletons such as the ones from Chen et al.  3.5 kg  2.9 kg , we estiFor clinical application of our exoskeleton the design and actuation profiles would need to be optimized. For example, the optimal timing could be different for every single patient as was found in stroke patients in Awad et al. . It is ubi-articular plus spring condition: closer to normal ankle and knee kinematics, additional assistance with knee flexion and higher total mechanical work assistance. However, we do not know to what extent each of these factors contributed to the metabolic cost result. Future exoskeleton designs could leverage each of these factors, possibly with different exoskeleton designs than ours, for example with separate ankle and knee actuation or with more lightweight and soft structures. Knowledge about specific effects of exoskeleton configuration on metabolic costs and muscle activation could be applied to providing customized assistance for different gait impairments or injuries and could also lead to novel experiments aimed at investigating the separate roles of the m. gastrocnemius and soleus.In conclusion, we found that a bi-articular exoskeleton configuration that mimics the m. gastrocnemius can reduce the metabolic cost of walking and reduce biceps femoris EMG. The following factors could have contributed to the higher reduction in metabolic rate in the PM, SG, WD, and DD designed the study. PM and SG performed data collection. PM performed the data analysis and drafted the manuscript. All authors edited and revised the analyses and the manuscript and gave final approval for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.