Facile de Novo Sequencing of Tetrazine-Cyclized Peptides through UV-Induced Ring-Opening and Cleavage from the Solid Phase.

While most FDA-approved peptide drugs are cyclic, the robust cyclization chemistry of peptides and the deconvolution of cyclic peptide sequences by using tandem mass spectrometry render cyclic peptide drug discovery difficult. Here we present the successful design of cyclic peptides on solid phase that addresses both of these problems. We demonstrate that this peptide cyclization method using dichloro-s-tetrazine on solid phase allows successful cyclization of a panel of random peptide sequences with various charges and hydrophobicities. The cyclic peptides can be linearized and cleaved from the solid phase by simple UV light irradiation, and we demonstrate that accurate sequence information can be obtained for the UV-cleaved linearized peptides by using tandem mass spectrometry. The tetrazine linker used in the cyclic peptides can further be explored for inverse electron-demand Diels-Alder (IEDDA) reactions for screening or bioconjugation applications in the future.

Mammary ductal elongation and myoepithelial migration are regulated by the composition of the extracellular matrix.

Mammary branching morphogenesis occurs over a period of weeks deep inside an adipocyte-rich stroma. The adipocytes contain light-scattering lipid droplets that limit the depth of penetration of visible light. Organotypic culture methods were developed to enable high-resolution optical monitoring of branching morphogenesis ex vivo. A challenge has been to identify the best culture conditions to model specific developmental events. We recently demonstrated that collagen I induces protrusive invasion in both normal and neoplastic mammary epithelium. In this study, we observed that the abundance of collagen I fibrils correlated strongly with invasive behaviour, even when the collagen I concentration was identical. We found that the extent of fibril assembly was experimentally manipulable by varying the incubation time at 4°C following pH neutralization. We next tested the capacity of collagen I fibrils to induce invasive behaviour when presented in combination with basement membrane proteins (Matrigel). We found that epithelial organoids in mixed gels of collagen I and basement membrane proteins exhibited more extensive branching morphogenesis but did not initiate protrusions into the matrix. Organoids in pure Matrigel produced many small epithelial buds that were bare of myoepithelial cells. Surprisingly, organoids in mixed gels of collagen I and Matrigel produced fewer epithelial buds, the buds elongated further, and the elongating buds remained covered by myoepithelial cells. Our mixed gels therefore provide a more physiologically accurate model of mammary branching morphogenesis. Our results also suggest that changes in the composition of the extracellular matrix could induce migration of epithelial cells past myoepithelial coverage.

Identification of novel mutations in BCKDHB and DBT genes in Vietnamese patients with maple sirup urine disease.

BACKGROUND: Maple sirup urine disease (MSUD) is an autosomal recessive inherited metabolic disorder. The disease-causing mutations can affect the BCKDHA, BCKDHB, and DBT genes encoding for the E1α, E1ß, and E2 subunits of the multienzyme branched-chain α-keto acid dehydrogenase (BCKDH) complex. In the present study, novel pathogenic variants in BCKDHB and DBT genes were identified in three Vietnamese families with MSUD. METHODS: Three newborn patients from three unrelated Vietnamese families were diagnosed with MSUD at the Metabolic Clinic, National Hospital of Pediatrics. Blood samples of 11 relatives from two generations of the three families diagnosed with MSUD were analyzed using exome and Sanger sequencing analyses. RESULTS: Novel pathogenic variants in BCKDHB (c.1103C>T, c.989A>G, and c.704G>A), and DBT (c.263_265delAAG) genes were identified in three pediatric patients with MSUD. CONCLUSIONS: We have identified novel pathogenic variants in the MSUD-related genes in the pedigree of the three patient's families. Our findings expand the mutational spectrum of MSUD and provide the scientific basis for genetic counseling for the patient's families.