Tsuyoshi Takato

Microtia is a congenital malformation of the auricle. The conventional therapy for microtia is reconstruction of the auricle by using the patient's own costal cartilage. Because it is invasive to harvest costal cartilages, less invasive ways for auricular reconstruction need to be established. Recent reports have indicated a new method for the production of cartilaginous particles from human induced pluripotent stem cells. To adopt this method to create an auricular-shaped regenerative cartilage, a novel scaffold with the property of a three-dimensional shape memory was created. A scaffold with a three-dimensional shape of auricular frames composed of a helix and an antihelix, which was designed to mimic an auricular framework carved from autologous costal cartilage and transplanted in auricular reconstruction, was prepared, filled with cartilaginous particles, and subcutaneously transplanted in nude rats. The auricular-shaped regenerative cartilage maintained the given shape and cartilage features in vivo for 1 year. Our findings suggest a novel approach for auricular reconstruction.

Contact domains of chromatin serve as a fundamental unit to regulate action of enhancers for target genes. Looping between a pair of CCCTC-binding factor (CTCF)-binding sites in convergent orientations underlies the formation of contact domains, while those in divergent orientations establish domain boundaries. However, every CTCF site is not necessarily engaged in loop or boundary structures, leaving functions of CTCF in varied genomic contexts still elusive. The locus containing Tfap2c and Bmp7 encompasses two contact domains separated by a region between the two genes, termed transition zone (TZ), characterized by two arrays of CTCF sites in divergent configuration. In this study, we created deletion and inversion alleles of these and other regions across the locus and investigated how they impinge on the conformation. Deletion of the whole two CTCF arrays with the CRISPR/Cas9 system resulted in impairment of blocking of chromatin contacts by the TZ, as assessed by the circular chromatin conformation capture assay (4C-seq). Deletion and inversion of either of the two arrays similarly, but less pronouncedly, led to reduction in the blocking activity. Thus, the divergent configuration provides the TZ with the strong boundary activity. Uniquely, we show the TZ harbors a 50-kb region within one of the two arrays that contacts broadly with the both flanking intervals, regardless of the presence or orientation of the other CTCF array. Further, we show the boundary CTCF array has little impact on intra-domain folding; instead, locally associating CTCF sites greatly affect it. Our results show that the TZ not only separates the two domains, but also bears a wide interval that shows isotropic behavior of chromatin folding, indicating a potentially complex nature of actual boundaries in the genome. We also show that CTCF-binding sites inside a domain greatly contribute to the intra-domain folding of chromatin. Thus, the study reveals diverse and context-dependent roles of CTCF in organizing chromatin conformation at different levels.

Background: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. The long-term complications arising from standard therapy with surgery, chemotherapy and radiotherapy lower the quality of life (QOL) due to eating disorders, speech problems and cosmetic issues. Recently the importance of antibody therapy has increasingly come to be recognized for its capacity to enhance QOL. Robo1, an axon guidance receptor, has been received considerable attention as a monoclonal antibody target in various cancers. Methods and findings: We checked the expression of Robo1 and the cytotoxic effect of saporin-conjugated anti- Robo1 immunotoxin (IT) against several HNSCC cell lines. The Robo1 cell surface expression level estimated by flow cytometry using the antibody B5209B was approximately 220,000 copies in Robo1 over-expressed CHO (Robo1/ CHO) cells, and in HNSCCs, 22,000 copies in HSQ-89 cells, 3,000 copies in Sa3 cells, and few copies inSAS cells. Conventional IT treatment exhibited an inadequate cytotoxic effect even in HSQ-89 cells. When we added a photosensitizer and LED light illumination (650 nm), the cytotoxic effect was remarkably improved in HSQ89. With longer exposure, significant cytotoxicity was observed, even in Sa3 cells. Conclusion: These results suggest that the photochemical internalization (PCI) is a promising method for augmenting the cytotoxicity of IT against tumors. The drug delivery system shown in this study should be applicable to other targets with low level expression on cancer cells, thus widening the therapeutic window in rare cancers.