Formation of AAB-Type Collagen Heterotrimers from Designed Cationic and Aromatic Collagen-Mimetic Peptides: Evaluation of the C-Terminal Cation-π Interactions.

Most of natural collagens are heterotrimers composed of two (AAB) or three (ABC) different peptide chains, and thus heterotrimeric constructs are preferable to mimic natural collagens. Exploring the forces to assemble synthetic collagen-mimetic peptides (CMPs) into heterotrimers has been an attractive topic in preparing collagen-related biomaterials. Here we designed and synthesized two cationic CMPs (CR and CK) in which multiple Arg or Lys residues are installed in their C-terminal region, and one aromatic CMP (CF) whose C-terminal end contains multiple Phe residues. Circular dichroism and NMR spectroscopy showed that AAB-type heterotrimers could form in both CR-CF and CK-CF mixtures, suggesting that the C-terminal cation-π interactions between cationic and aromatic residues could serve as a nucleation force and substantially promote the folding of heterotrimers. In particular, only one major heterotrimeric fold was found in each mixture. For CR-CF mixtures, either the heterotrimer with two CR chains and one CF chain or that with one CR chain and two CF chains could form, depending on the molar ratios of CR to CF in solution. By contrast, in CK-CF mixtures only the heterotrimer consisting of two CK chains and one CF chain was found in solution even increasing the ratio of CF, implying that the heterotrimer composed of one CK chain and two CF chains is highly unstable. Additionally, differential scanning calorimetry analysis showed that the folding of these heterotrimers is governed by entropic effects. Together, our results provide a new design to prepare AAB-type collagen heterotrimers and reveal new insights into their folding thermodynamics.

Hepatic stellate cells promote the differentiation of embryonic stem cell-derived definitive endodermal cells into hepatic progenitor cells.

AIM: Hepatic non-parenchymal cells are well known to be capable of providing an important microenvironment and growth factors for hepatic regeneration, but their capacity for directing embryonic stem cells (ESC) toward hepatocytes remains to be assessed. Thus, this study aims to investigate the role of hepatic stellate cells (HSC), the major type of hepatic non-parenchymal cells, in the differentiation of ESC as well as exploring the potentiality of ESC in regeneration medicine for cell-based therapy. METHODS: A two-step differentiation procedure that utilized the capability of HSC to regulate proliferation and differentiation of hepatocytes was used to develop an approach for directing the differentiation of ESC towards hepatic progenitor cells. Mouse ESC were cultivated in a serum-free medium containing Activin A and fibroblast growth factor to generate definitive endodermal cells characterized by the CXCR4 cell-surface marker. After 6-8 days in culture, approximately 60% of the differentiated cells expressed CXCR4, and more than 90% of the CXCR4 positive cells could be recovered by cell sorting. The purified CXCR4 positive cells were co-cultured with mouse HSC as feeder cells in basal medium without additional hepatocyte growth factors. Differentiation was complete after 10-12 days of co-culture, and hepatic progenitor cell markers such as α-fetoprotein (afp) and albumin (alb) were detected in the terminally differentiated ESC. CONCLUSION: These results show that HSC provide an appropriate microenvironment and pivotal growth factors for generation of hepatic progenitor cells from ESC-derived definitive endodermal cells, and suggest that this approach possibly allows for hepatic differentiation of ESC imitating the process of hepatic regeneration.

Synthesis, Isolation, and Characterization of Mono- and Bis-norbornene-Annulated Biarylamines through Pseudo-Catellani Intermediates.

A palladium-catalyzed C-H functionalization of an external ring of N-acyl 2-aminobiaryl with bicyclo[2.2.1]hept-2-ene (norbornene) via multiple C-H bond activations was developed. This study is the first report of the formation of bis-norbornene annulated biarylamines isomers ( syn-3a'/ anti-3a' = 36:64) from multiple C-H bond functionalizations. Additionally, nondirected C-H bond functionalization at the C-4' position with alkenes rendered complete C-H functionalization of five C-H bonds that formed a stable hexasubstituted benzene ring.