The epithelial-mesenchymal transition (EMT) is critical for induction of invasiveness and metastasis of human cancers. In this study we investigated the expression profiles of the EMT markers, the relationship between EMT markers and patient/tumor/viral factors, and the interplay between major EMT regulators in human hepatocellular carcinoma (HCC). Reduced E-cadherin and nonmembranous ␤-catenin expression, the hallmarks of EMT, were shown in 60.2% and 51.5% of primary HCC samples, respectively. Overexpression of Snail, Twist, or Slug, the major regulators of EMT, was identified in 56.9%, 43.1%, and 51.4% of primary HCCs, respectively. Statistical analysis determined that Snail and Twist, but not Slug, are major EMT inducers in HCC: overexpression of Snail and/or Twist correlated with down-regulation of E-cadherin, nonmembranous expression of ␤-catenin, and a worse prognosis. In contrast, there were no such significant differences in samples that overexpressed Slug. Coexpression of Snail and Twist correlated with the worst prognosis of HCC. Hepatitis Cassociated HCC was significantly correlated with Twist overexpression. HCC cell lines with increased Snail and Twist expression (e.g., Mahlavu) exhibited a greater capacity for invasiveness/metastasis than cells with low endogenous Twist/Snail expression (e.g., Huh-7). Overexpression of Snail or/and Twist in Huh-7 induced EMT and invasiveness/metastasis, whereas knockdown of Twist or Snail in Mahlavu reversed EMT and inhibited invasiveness/metastasis. Twist and Snail were independently regulated, but exerted an additive inhibitory effect to suppress E-cadherin transcription. Conclusion: Our study provides a comprehensive profile of EMT markers in HCC, and the independent and collaborative effects of Snail and Twist on HCC metastasis were confirmed through different assays.

Traditional interpenetrating polymer networks (IPNs) are not adaptable materials because the topological structure of the macromolecules cannot be changed, which limits their structural rearrangement, reprocessing, and recycling. Here in this work we present a strategy for preparing reversibly interlocking networks (RILNs) from two preformed immiscible polymer networks based on dynamic covalent chemistry. The frequently opening and closing of the single networks enabled by the exchange reactions of the embedded orthogonal dynamic covalent bonds and stronger intercomponent interaction mainly account for the formation of the interlocking topology architecture of the RILNs. The resultant RILNs are rather homogeneous, which not only possess stimulus-responsive adaptive performance like self-healing but also exhibit nonlinear improvement in static and dynamic mechanical properties. By taking advantage of the reversible bonding, more importantly, the RILNs can be unlocked reproducing the pristine single networks, and the relocking/unlocking cycling is allowed to proceed for multiple times, which are not available for IPNs as defined by their chemical nature. It is anticipated that the proposed methodology provides a new idea for producing multifunctional cross-linked polymers capable of repeated controlled degradation and regeneration.

Cbln1 is a secreted glycoprotein essential for synapse structure and function in cerebellum that is also expressed in extracerebellar structures where its function is unknown. Furthermore, Cbln1 assembles into homomeric complexes and heteromeric complexes with three family members (Cbln2-Cbln4), thereby influencing each other's degradation and secretion. Therefore, to understand its function, it is essential to establish the location of Cbln1 relative to other family members. The localization of Cbln1 in brain was determined using immunohistochemistry and cbln1-lacZ transgenic mice. Cbln1-like immunoreactivity (CLI) was always punctate and localized to the cytoplasm of neurons. The punctate CLI colocalized with cathepsin D, a lysosomal marker, but not with markers of endoplasmic reticulum or Golgi, indicating that Cbln1 is present in neuronal endosomes/lysosomes. This may represent the cellular mechanism underlying the regulated degradation of Cbln1 observed in vivo. Outside the cerebellum, CLI mapped to multiple brain regions that were frequently synaptically interconnected, warranting their analysis in cbln1-null mice. Furthermore, whereas CLI increased dramatically in the cerebellum of cbln3-null mice it was unchanged in extracerebellar neurons. This opens the possibility that other family members that are coexpressed in these areas control Cbln1 levels, potentially by modulating processing in the endolysosomal pathway. During development of cbln1-lacZ mice, beta-galactosidase staining was first observed in proliferating granule cell precursors prior to synaptogenesis and thereafter in maturing and adult granule cells. As cbln3 is only expressed in post-mitotic, post-migratory granule cells, Cbln1 homomeric complexes in precursors and Cbln1-Cbln3 heteromeric complexes in mature granule cells may have distinct functions and turnover.

Cbln1 is essential for synapse integrity in cerebellum through assembly into complexes that bridge presynaptic β-neurexins (Nrxn) to postsynaptic GluRδ2. However, GluRδ2 is largely cerebellum-specific, yet Cbln1 and its little studied family members, Cbln2 and Cbln4, are expressed throughout brain. Therefore, we investigated whether additional proteins mediate Cbln family actions. Whereas Cbln1 and Cbln2 bound to GluRδ2 and Nrxns1–3, Cbln4 bound weakly or not at all, suggesting it has distinct binding partners. In a candidate receptor-screening assay, Cbln4 (but not Cbln1 or Cbln2) bound selectively to the netrin receptor, DCC (deleted in colorectal cancer) in a netrin-displaceable fashion. To determine whether Cbln4 had a netrin-like function, Cbln4-null mice were generated. Cbln4-null mice did not phenocopy netrin-null mice. Cbln1 and Cbln4 were likely co-localized in neurons thought to be responsible for synaptic changes in striatum of Cbln1-null mice. Furthermore, complexes containing Cbln1 and Cbln4 had greatly reduced affinity to DCC but increased affinity to Nrxns, suggesting a functional interaction. However, Cbln4-null mice lacked the striatal synaptic changes seen in Cbln1-null mice. Thus Cbln family members interact with multiple receptors/signaling pathways in a subunit composition-dependent manner and have independent functions with Cbln4 potentially involved in the less-well characterized role of netrin/DCC in adult brain.

Neuroligins are postsynaptic cell-adhesion molecules that bind to presynaptic neurexins. Mutations in neuroligin-3 predispose to autism, but how such mutations affect synaptic function remains incompletely understood. Here we systematically examined the effect of three autism-associated mutations, the neuroligin-3 knockout, the R451C knockin, and the R704C knockin, on synaptic transmission in the calyx of Held, a central synapse ideally suited for high-resolution analyses of synaptic transmission. Surprisingly, germline knockout of neuroligin-3 did not alter synaptic transmission, whereas the neuroligin-3 R451C and R704C knockins decreased and increased, respectively, synaptic transmission. These puzzling results prompted us to ask whether neuroligin-3 mutant phenotypes may be reshaped by developmental plasticity. Indeed, conditional knockout of neuroligin-3 during late development produced a marked synaptic phenotype, whereas conditional knockout of neuroligin-3 during early development caused no detectable effect, mimicking the germline knockout. In canvassing potentially redundant candidate genes, we identified developmentally early expression of another synaptic neurexin ligand, cerebellin-1. Strikingly, developmentally early conditional knockout of cerebellin-1 only modestly impaired synaptic transmission, whereas in contrast to the individual single knockouts, developmentally early conditional double knockout of both cerebellin-1 and neuroligin-3 severely decreased synaptic transmission. Our data suggest an unanticipated mechanism of developmental compensation whereby cerebellin-1 and neuroligin-3 functionally occlude each other during development of calyx synapses. Thus, although acute manipulations more likely reveal basic gene functions, developmental plasticity can be a major factor in shaping the overall phenotypes of genetic neuropsychiatric disorders.