Sox9 is a direct transcriptional activator of cartilage-specific extracellular matrix genes and has essential roles in chondrogenesis. Mutations in or around the SOX9 gene cause campomelic dysplasia or Pierre Robin Sequence. However, Sox9-dependent transcriptional control in chondrogenesis remains largely unknown. Here we identify Wwp2 as a direct target of Sox9. Wwp2 interacts physically with Sox9 and is associated with Sox9 transcriptional activity via its nuclear translocation. A yeast two-hybrid screen using a cDNA library reveals that Wwp2 interacts with Med25, a component of the Mediator complex. The positive regulation of Sox9 transcriptional activity by Wwp2 is mediated by the binding between Sox9 and Med25. In zebrafish, morpholino-mediated knockdown of either wwp2 or med25 induces palatal malformation, which is comparable to that in sox9 mutants. These results provide evidence that the regulatory interaction between Sox9, Wwp2 and Med25 defines the Sox9 transcriptional mechanisms of chondrogenesis in the forming palate.
Human embryonic stem (ES) cells have the potential to differentiate into all cell types. As these cells may be able to provide an unlimited cell source for transplantation therapies, it is necessary to establish reliable methods for their handling and manipulation, including human ES cell cryopreservation. Here, we report the development of a simple and efficient cryopreservation method for primate ES cell lines using vitrification in conventional cryovials. Using standard slowrate cooling methods, the cryopreservation efficiency for cynomolgus monkey ES cell lines was approximately 0.4%, while that for a human ES cell line was virtually 0%. Primate ES cell lines, however, were successfully cryopreserved by the present vitrification method using conventional cryovials yielding a survival rate of about 6.5% for monkey ES cells and 12.2% for human ES cells. Vitrified ES cells quickly recovered after thawing and exhibited a morphology indistinguishable from non-vitrified cells. In addition, they retained a normal karyotype and continued to express ES cell markers after thawing. Thus, our vitrification ES cell cryopreservation method expands the utility of primate ES cells for various research and clinical purposes.
MicroRNA-140 (miR-140) is Specifically expressed in developing cartilage tissues. We have previously reported that miR-140 plays an important role during palatal cartilage development by modulating platalet-derived growth factor receptor alpha (pdgfra) in zebrafish. However, the regulatory mechanism of miR-140 in cartilage is still unknown. Using developing zebrafish, Sox9a mutant (Sox9a−/−) and Sox9b mutant (Sox9b−/−) zebrafish and Sox9 siRNA in human chondrocytes, T/C-28 Cells, we found that miR-140 is regulated by the cartilage master transcription regulator Sox9 in zebrafish and mammalian cells.
The factors related to poor outcomes were, age of 60 years or over, pre-operative Hunt & Kosnik grade II or more, Fisher Scale 3 or more, aneurysm size over 15 mm in diameter, and location at and around the basilar artery bifurcation. The results presented in this study show the status of our direct surgical management of subarachnoid haemorrhage in a large series with standardized surgical principles and procedures.
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