Biological evaluation of human degenerated nucleus pulposus cells in functionalized self-assembling peptide nanofiber hydrogel scaffold.

Nucleus pulposus (NP) tissue engineering has been proposed as a novel biological treatment for early-stage intervertebral disc degeneration. In this study, a novel functional self-assembling peptide PKP was first designed by linking the short functional motif of bone morphogenetic protein-7 (BMP7) to the C-terminal of RADA16-I, and another new functional self-assembling peptide was obtained by mixing RKP with RADA16-I. Then, the biocompatibilities and bioactivities of RKP and RAD-RKP for human degenerated nucleus pulposus cells (hNPCs) were studied in vitro. Atomic force microscopy and scanning electron microscopy (SEM) confirmed that both RKP and RAD-RKP could self-assemble into three-dimensional (3D) nanofiber hydrogel scaffolds in a culture medium at 37°C. After the hNPCs were cultured in 3D scaffolds, both RKP and RAD-RKP exhibited reliable attachment and extremely low cytotoxicities (<14%), which were verified by SEM and cytotoxity assays, respectively. Our results also showed that the functional-based scaffolds could increase the proliferation and migration of hNPCs after 7 days compared with culture plates and pure RADA16-I. Quantitative real-time polymerase chain reaction demonstrated that the expressions of collagen II α1, Sox-9, and aggrecan were upregulated, while collagen I α1 was downregulated by functional-based scaffolds after 28 days. Furthermore, we also confirmed that RAD-RKP exhibited a higher hNPC proliferation, migration, and expression of Sox-9 and aggrecan compared with pure RKP. Therefore, the results of this study indicated that the BMP7 short motif-designed functional self-assembling peptide nanofiber hydrogels could be used as excellent scaffolds in NP tissue engineering, and RAD-RKP might have further potential application in human mild degenerated NP tissue regeneration.

Preparation of magnetic polymer material with phosphate group and its application to the enrichment of phosphopeptides.

As one of the most important post-translational modifications (PTM), reversible phosphorylation of protein is involved in many cellular processes. Enrichment and separation of phosphopeptides have become essential for large-scale identification of protein phosphorylation by mass spectrometry. In this work, five magnetic polymer materials with different numbers of phosphate groups were fabricated using a simple polymeric method and their abilities to enrich phosphopeptides were investigated. Our results showed that the enrichment efficiency is closely related to the number of phosphate groups attached to magnetic polymer sorbent. Under optimized condition (3% trifluoroacetic acid and 80% acetonitrile), magnetic polymer-particles with appropriate proportion of phosphate groups (Fe(3)O(4)@p(VPA-EDMA-1)-Zr(4+)) showed high performance for extracting phosphopeptides from complex peptides mixture of standard protein digestion. In this regard, a total of 988 unique phosphopeptides were successfully identified from proteolytic digestion of HeLa cell extracts by employing magnetic polymer-particles combined with nano-RPLC-MS/MS analysis.

Displacement of D1, HP1 and topoisomerase II from satellite heterochromatin by a specific polyamide.

The functions of DNA satellites of centric heterochromatin are difficult to assess with classical molecular biology tools. Using a chemical approach, we demonstrate that synthetic polyamides that specifically target AT-rich satellite repeats of Drosophila melanogaster can be used to study the function of these sequences. The P9 polyamide, which binds the X-chromosome 1.688 g/cm3 satellite III (SAT III), displaces the D1 protein. This displacement in turn results in a selective loss of HP1 and topoisomerase II from SAT III, while these proteins remain bound to the adjacent rDNA repeats and to other regions not targeted by P9. Conversely, targeting of (AAGAG)n satellite V repeats by the P31 polyamide results in the displacement of HP1 from these sequences, indicating that HP1 interactions with chromatin are sensitive to DNA-binding ligands. P9 fed to larvae suppresses the position-effect variegation phenotype of white-mottled adult flies. We propose that this effect is due to displacement of the heterochromatin proteins D1, HP1 and topoisomerase II from SAT III, hence resulting in stochastic chromatin opening and desilencing of the nearby white gene.