Osteoblastic differentiation on hydrogels fabricated from Ca2+-responsive self-assembling peptides functionalized with bioactive peptides.

We recently developed an amphiphilic peptide, E1Y9 (Ac-E-YEYKYEYKY-NH2), that self-assembles into nanofibers and forms a hydrogel in the presence of Ca2+ ion. Four E1Y9-derivatives (E1Y9-ALK, E1Y9-DGR, E1Y9-PRG and E1Y9-RGD) were designed as conjugates of E1Y9 with bioactive peptide sequences named as ALK (ALKRQGRTLYGF), DGR (DGRDSVAYG), PRG (PRGDSGYRGDS) and RGD (RGDS), respectively, and stimulated osteoblast cells growth as well as differentiation. In this study, E1Y9/E1Y9-derivative mixed hydrogels were constructed to serve as scaffolds for osteoblastic differentiation of MC3T3-E1 cells. E1Y9 and E1Y9-derivatives co-assembled into networked nanofibers and formed hydrogels in response to Ca2+ ion. The pre-osteoblast cell line MC3T3-E1 was cultured and differentiated on mixed hydrogels. An E1Y9/E1Y9-ALK mixed hydrogel exhibited the highest cell proliferation and differentiation activity among the peptide hydrogels. The peptide sequence ALK promoted expression of RUNX2 and osteopontin, a key transcription factor and bone tissue matrix protein, respectively, during the differentiation stage. During the later stage, localization of RUNX2 and osteopontin was regulated in the cytosol and extracellularly, respectively, indicating that the E1Y9/E1Y9-ALK mixed hydrogel controlled the differentiation of MC3T3-E1 cells. Thus, the E1Y9/E1Y9-ALK mixed hydrogel developed in this study showed potential for the culture and regulation of differentiation of osteoblast cells for bone regeneration.

Characterization of Shiga toxin-producing Escherichia coli from feces of sika deer (Cervus nippon) in Japan using PCR binary typing analysis to evaluate their potential human pathogenicity.

This study examined the potential pathogenicity of Shiga toxin-producing Escherichia coli (STEC) in feces of sika deer by PCR binary typing (P-BIT), using 24 selected STEC genes. A total of 31 STEC strains derived from sika deer in 6 prefectures of Japan were O-serotyped and found to be O93 (n=12), O146 (n=5), O176 (n=3), O130 (n=3), O5 (n=2), O7 (n=1), O96 (n=1), O116 (n=1), O141 (n=1), O157 (n=1) and O-untypable (n=1). Of the 31 STEC strains, 13 carried both stx1 and stx2, 5 carried only stx1, and 13 carried one or two variants of stx2. However, no Stx2 production was observed in 3 strains that carried only stx2: the other 28 strains produced the appropriate Stx. P-BIT analysis showed that the 5 O5 strains from two wild deer formed a cluster with human STEC strains, suggesting that the profiles of the presence of the 24 P-BIT genes in the deer strains were significantly similar to those in human strains. All of the other non-O157 STEC strains in this study were classified with strains from food, domestic animals and humans in another cluster. Good sanitary conditions should be used for deer meat processing to avoid STEC contamination, because STEC is prevalent in deer and deer may be a potential source of STEC causing human infections.

Structural development of stapled short helical peptides as vitamin D receptor (VDR)-coactivator interaction inhibitors.

We developed several stabilized helical heptapeptides (DPI-01-10) composed of l-leucine residues, an α,α-disubstituted α-amino acid (α-aminoisobutyric acid [Aib] or hydroxymethylserine [Hms]), and a stapled side chain as inhibitors of vitamin D receptor (VDR)-coactivator interactions. The inhibitory activity of these peptides against VDR-coactivator interactions was evaluated using a receptor cofactor assay system, and DPI-08 demonstrated strong activity (IC50: 3.2µM).

Development of stapled short helical peptides capable of inhibiting vitamin D receptor (VDR)-coactivator interactions.

We synthesized stapled helical leucine-based peptides (DPI-01-07) containing 2-aminoisobutyric acid and a covalent cross-linked unit as inhibitors of vitamin D receptor (VDR)-coactivator interactions. The effects of these peptides on the human VDR were examined in an inhibition assay based on the receptor cofactor assay system, and one of them, DPI-07, exhibited potent inhibitory activity (IC50: 3.2 µM).