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The ubiquitin-like modifier (UBL) family has recently generated much interest in the scientific community, as it is implicated to play important regulatory roles via novel protein-protein modification. FAT10 (diubiquitin) belongs to this family of proteins, comprising two ubiquitin-like moieties fused in tandem, and has been implicated to be involved in the maintenance of spindle integrity during mitosis. As FAT10 may play a role in the regulation of genomic stability, we examined if there is an association between FAT10 expression and hepatocellular carcinoma (HCC) or other cancers. Northern blot analyses revealed upregulation of FAT10 expression in the tumors of 90% of HCC patients. In situ hybridization as well as immunohistochemistry utilizing anti-FAT10 antibodies localized highest FAT10 expression in the nucleus of HCC hepatocytes rather than the surrounding immune and non-HCC cells. FAT10 expression was also found to be highly upregulated in other cancers of the gastrointestinal tract and female reproductive system. In conclusion, we demonstrated upregulation of FAT10 expression in various gastrointestinal and gynecological cancers. Its overexpression is unrelated to the general increase in protein synthesis or a general immune/ inflammatory response to cancer. Rather, FAT10 may modulate tumorigenesis through its reported interaction with the MAD2 spindle-assembly checkpoint protein.

SignificanceMimicking protein-like specific interactions and functions has been a long-pursued goal in nanotechnology. The key challenge is to precisely organize nonfunctional surface groups on nanoparticles into specific 3D conformations to function in a concerted and orchestrated manner. Here, we develop a method to graft the complementary-determining regions of natural antibodies onto nanoparticles and reconstruct their “active” conformation to create nanoparticle-based artificial antibodies that recognize the corresponding antigens. Our work demonstrates that it is possible to create functions on nanoparticles by conformational engineering, namely tuning flexible surface groups into specific conformations. Our straightforward strategy could be used further to create other artificial antibodies for various applications and provides a new tool to understand the structure and folding of natural proteins.

Metallothioneins (MTs) belong to a family of cysteine-rich, metal-binding intracellular proteins, which have been linked with cell proliferation. In this study, expression levels of the 8 known MT-1 and MT-2 functional isoforms in human invasive ductal breast cancer specimens were determined by RT-PCR. The expression profiles of the MT protein and MT-2A mRNA were further evaluated in 79 cases of human invasive ductal breast carcinoma by immunohistochemistry and in situ hybridization, and correlated with cancer cell proliferation (determined by Ki-67 nuclear antigen immunolabeling). MT-1A, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X and MT-2A but not MT-1B, were detected in breast cancer tissue samples. The MT-2A mRNA transcript was the highest among all the isoforms detected. A positive correlation was observed between MT-2A mRNA and MT protein expression with Ki-67 labeling (P = 0.0003 and P < 0.0001, respectively) but not with apoptosis (P = 0.1244 and P = 0.8189, respectively). Co-localization of the MT protein and Ki-67 nuclear antigen in breast cancer cells was demonstrated by double immunofluorescence staining. There was also significantly higher MT protein and MT-2A mRNA expression in histological grade 3 tumors than in histological grade 1 and 2 tumors. The finding that MT 2A appears to be the main isoform associated with cell proliferation in invasive ductal breast cancer tissues, may have therapeutic implications.

Enzymatic crosslinking of dextran-tyramine (Dex-TA) conjugates in the presence of horseradish peroxidase and hydrogen peroxide was successively applied in the preparation of hydrogels. Depending on the molecular weight of the dextran (M(n,) (GPC) of 14000 or 31000 g/mol) and the degree of substitution (of 5, 10, or 15) with TA groups, the gelation times ranged from 20 s to 1 min. Hydrogels prepared from Dex31k-TA with a degree of substitution of 10 had storage moduli up to 60 kPa. Similar values were found when chondrocytes were incorporated into the hydrogels. Chondrocyte-seeded Dex-TA hydrogels were prepared at a molar ratio of hydrogen peroxide/TA of 0.2 and cultured in a chondrocyte medium. A live-dead assay and a methylthiazol tetrazolium assay revealed that almost all chondrocytes retained their viability after 2 weeks. Scanning electron microscopy analysis showed that the encapsulated chondrocytes were capable of maintaining their round shape. Histology and immunofluorescent staining demonstrated the production of glycosaminoglycans (GAGs) and collagen type II after culturing for 14 and 21 days. Biochemical analysis showed that GAG accumulation increased with time inside Dex-TA hydrogels. Besides, GAG/DNA for Dex-TA hydrogels was higher than that for agarose at day 28. These results indicate that Dex-TA hydrogels are promising 3D scaffolds for cartilage tissue engineering applications.