Dual-targeted nano-encapsulation of neonatal porcine islet-like cell clusters with triiodothyronine-loaded bifunctional polymersomes.

There is growing evidence that neonatal porcine islet-like cell clusters (NPCCs) isolated from piglets can be used to treat type 1 diabetes in humans. However, graft rejection is a common complication in humans owing to the prevalence of xenoantigens in porcine. Therefore, researchers have investigated various islet encapsulation techniques that could protect against these antigens. To this end, this study presents a robust nano-encapsulation method based on bifunctional polymersomes (PSomes), in which N-hydroxysuccinimide (NHS) and maleimide (Mal) groups conjugated to the PSomes terminal interact with the amine and thiol groups on the surface of NPCCs to induce dual targeting via two covalent bonds. The findings indicate that the ratio of NHS to Mal on PSomes is optimal for dual targeting. Moreover, triiodothyronine (T3) is known to promotes pancreatic islet maturation and differentiation of endocrine cells into beta cells. T3 encapsulated in PSomes is shown to increase the glucose sensitivity of NPCCs and enhance insulin secretion from NPCCs. Furthermore, improvements in the nano-encapsulation efficiency and insulin-secreting capability of NPCCs through dual targeting via dual-Psomes are demonstrated. In conclusion, the proposed nano-encapsulation technique could pave the way for significant advances in islet nano-encapsulation and the imprevement of NPCC immaturity via T3 release.

Production of genetically modified pigs expressing human insulin and C-peptide as a source of islets for xenotransplantation.

Islet xenotransplantation is a promising treatment for type I diabetes. Numerous studies of islet xenotransplantation have used pig-to-nonhuman primate transplantation models. Some studies reported long-term survival and successful function of porcine islets in diabetic monkeys. Genetic engineering techniques may improve the survival and function of porcine islets. A recent study reported the generation of transgenic pigs expressing human insulin rather than porcine insulin by changing one amino acid at the end of the ß-chain in insulin. However, C-peptide from pigs still existed. In this study, we generated transgenic pigs expressing human proinsulin to express human insulin and C-peptide using fibroblasts from proinsulin knockout pigs as donor cells for somatic cell nuclear transfer. Eleven live piglets were delivered from three surrogates and characterized to confirm the genotype and phenotype of the generated piglets. Genotype analysis of the generated piglets showed that five of the eleven piglets contained the human proinsulin gene. Insulin expression was confirmed in the serum and pancreas in two of the five piglets. C-peptide derived from human proinsulin was also confirmed by liquid chromatography tandem mass spectrometry. Non-fasting blood glucose level was measured to verify the function of the insulin derived from the human proinsulin. Two piglets expressing insulin showed normal glucose levels similar to that in the wild-type control. In conclusion, human insulin- and C-peptide-expressing pigs without porcine insulin and C-peptide were successfully established. These pigs can be used as a source of islets for islet xenotransplantation.

Generation of insulin-deficient piglets by disrupting INS gene using CRISPR/Cas9 system.

Diabetes mellitus is a chronic disease with accompanying severe complications. Various animal models, mostly rodents due to availability of genetically modified lines, have been used to investigate the pathophysiology of diabetes. Using pigs for diabetic research can be beneficial because of their similarity in size, pathogenesis pathway, physiology, and metabolism with human. However, the use of pigs for diabetes research has been hampered due to only few pig models presenting diabetes symptoms. In this study, we have successfully generated insulin-deficient pigs by generating the indels of the porcine INS gene in somatic cells using CRISPR/Cas9 system followed by somatic cell nuclear transfer. First, somatic cells carrying a modified INS gene were generated using CRISPR/Cas9 system and their genotypes were confirmed by T7E1 assay; targeting efficiency was 40.4% (21/52). After embryo transfer, three live and five stillborn piglets were born. As expected, INS knockout piglets presented high blood glucose levels and glucose was detected in the urine. The level of insulin and c-peptide in the blood serum of INS knockout piglets were constant after feeding and the expression of insulin in the pancreas was absent in those piglets. This study demonstrates effectiveness of CRISPR/Cas9 system in generating novel pig models. We expect that these insulin-deficient pigs can be used in diabetes research to test the efficacy and safety of new drugs and the recipient of islet transplantation to investigate optimal transplantation strategies.

Clinical, genomic, and metagenomic characterization of oral tongue squamous cell carcinoma in patients who do not smoke.

BACKGROUND: Evidence suggests the incidence of oral tongue squamous cell carcinoma is increasing in young patients, many who have no history of tobacco use. METHODS: We clinically reviewed 89 patients with oral tongue cancer. Exomic sequencing of tumor DNA from 6 nonsmokers was performed and compared to previously sequenced cases. RNA from 20 tumors was evaluated by massively parallel sequencing to search for potentially oncogenic viruses. RESULTS: Non-smokers (53 of 89) were younger than smokers (36 of 89; mean, 50.4 vs 61.9 years; p < .001), and seemed more likely to be women (58.5% vs 38.9%; p = .069). Nonsmokers had fewer TP53 mutations (p = .02) than smokers. No tumor-associated viruses were detected. CONCLUSION: The young age of nonsmoking patients with oral tongue cancer and fewer TP53 mutations suggest a viral role in this disease. Our efforts to identify such a virus were unsuccessful. Further studies are warranted to elucidate the drivers of carcinogenesis in these patients.

Expression microarray analysis reveals alternative splicing of LAMA3 and DST genes in head and neck squamous cell carcinoma.

PURPOSE: Prior studies have demonstrated tumor-specific alternative splicing events in various solid tumor types. The role of alternative splicing in the development and progression of head and neck squamous cell carcinoma (HNSCC) is unclear. Our study queried exon-level expression to implicate splice variants in HNSCC tumors. EXPERIMENTAL DESIGN: We performed a comparative genome-wide analysis of 44 HNSCC tumors and 25 uvulopalatopharyngoplasty (UPPP) tissue samples at an exon expression level. In our comparison we ranked genes based upon a novel score-the Maximum-Minimum Exon Score (MMES)--designed to predict the likelihood of an alternative splicing event occurring. We validated predicted alternative splicing events using quantitative RT-PCR on an independent cohort. RESULTS: After MMES scoring of 17,422 genes, the top 900 genes with the highest scores underwent additional manual inspection of expression patterns in a graphical analysis. The genes LAMA3, DST, VEGFC, SDHA, RASIP1, and TP63 were selected for further validation studies because of a high frequency of alternative splicing suggested in our graphical analysis, and literature review showing their biological relevance and known splicing patterns. We confirmed TP63 as having dominant expression of the short DeltaNp63 isoform in HNSCC tumor samples, consistent with prior reports. Two of the six genes (LAMA3 and DST) validated by quantitative RT-PCR for tumor-specific alternative splicing events (Student's t test, P<0.001). CONCLUSION: Alternative splicing events of oncologically relevant proteins occur in HNSCC. The number of genes expressing tumor-specific splice variants needs further elucidation, as does the functional significance of selective isoform expression.