Identification and analysis of a selective DYRK1A inhibitor.

The dysregulation of DYRK1A is implicated in many diseases such as cancer, diabetes, and neurodegenerative diseases. Alzheimer's disease is one of the most common neurodegenerative disease and has elevated interest in DYRK1A research. Overexpression of DYRK1A has been linked to the formation of tau aggregates. Currently, an effective therapeutic treatment that targets DYRK1A is lacking. A specific small-molecule inhibitor would further our understanding of the physiological role of DYRK1A in neurodegenerative diseases and could be presented as a possible therapeutic option. In this study, we identified pharmacological interactions within the DYRK1A active site and performed a structure-based virtual screening approach to identify a selective small-molecule inhibitor. Several compounds were selected in silico for enzymatic and cellular assays, yielding a novel inhibitor. A structure-activity relationship analysis was performed to identify areas of interactions for the compounds selected in this study. When tested in vitro, reduction of DYRK1A dependent phosphorylation of tau was observed for active compounds. The active compounds also improved tau turbidity, suggesting that these compounds could alleviate aberrant tau aggregation. Testing the active compound against a panel of kinases across the kinome revealed greater selectivity towards DYRK1A. Our study demonstrates a serviceable protocol that identified a novel and selective DYRK1A inhibitor with potential for further study in tau-related pathologies.

Nanofibers grafted on titanium alloy: the effects of fiber alignment and density on osteoblast mineralization.

The surface of medical implant alloy Ti-6Al-4V was chemically modified to allow it to covalently bond with collagen/PVA nanofibers. These nanofibers were successfully attached to the Ti-6Al-4V surface in three different morphologies: randomly oriented high-density fiber, COL(H); randomly oriented low-density fiber, COL(L); and aligned high-density fiber, COL(A). The effects of the morphology of these covalently-bound collagen nanofibers on the growth and differentiation of osteoblasts were studied for 21 days. The low-density nanofibers covered approximately 80% of the Ti64 surface, while the high-density nanofibers covered nearly 100%. These covalently attached fibrous coatings remained attached to the metal surface after 3 weeks of cell culture. In the first week the aligned fibers of COL(A) allowed the osteoblasts to stretch and elongate in the direction of the fibers. This directional elongation was not seen in the cells on the randomly-oriented samples. Cells proliferated and differentiated on all three surfaces over time. By the end of the test, the amount of type I collagen secreted by the cells on COL(H) was the highest, while the degree of mineralization was highest on COL(A) among the three samples (p < 0.05). Different nanofiber morphologies changed the cell morphology and the secretion of cellular products. The mechanisms remained to be investigated. The surface of medical implant alloy Ti-6Al-4V was chemically modified to allow it to covalently bond with collagen/PVA nanofibers. The SEM micrographs in the top row show the random and aligned morphology of the collagen-PVA nanofibers. The nanofibers on COL(A) were aligned in the general direction indicated by the arrow. The second row are images from EDX titanium element mapping. The location of the titanium elements are shown as bright dots. The low-density nanofibers, COL(L), covered approximately 80% of the Ti64 surface, while the high-density nanofibers, COL(H) and COL(A), covered nearly 100%. All three surfaces demonstrated good biocompatibility for the cultured osteoblasts. The fiber alignment seemed to have an effect on early cellular morphology (day 7), collagen secretion and calcium deposition, while the density of the fibers seemed to have no significant effect on cell behavior. SEM micrographs of osteoblasts after 7 and 14 days of cell culture are shown in the third and fourth rows. The surface of COL(L) has more cell-free spots indicated by (*) on day 7 as other two surfaces were covered by cells. The nanofibers could no longer be observed and were covered with mineralized granules (circles) after 14 days of cell culture. The cells appear stretched out on the mineralized granules.

Chondrosarcoma of the proximal humerus secondary to ollier disease: an 8-year follow-up of successful resection of the tumor with endoprosthetic replacement of the proximal humerus.

We present a 25-year-old male patient with a diagnosis of multiple enchondromatosis, who developed chondrosarcoma on the proximal humerus of the right upper limb. The patient had the pre-existing lesions of Ollier's disease discovered during his childhood. The patient underwent wide resection of the sarcoma with a prosthetic replacement of the proximal humerus. So far we have followed up the patient for 8 years with no evidence of local recurrence and/or metastasis. The therapeutic results have been satisfied with a good functional recovery of the treated limb, enabling the patient to return to the pre-disease daily living and occupational activities. The reconstructive procedures represent an effective surgical strategy for limb salvage in the treatment of large segmental defects after resection of humeral tumors, substantially solving the functional and esthetic problems due to such a wide resection, and significantly improving the quality of life for the patient.

Osteosarcoma cells promote the production of pro-tumor cytokines in mesenchymal stem cells by inhibiting their osteogenic differentiation through the TGF-ß/Smad2/3 pathway.

Mesenchymal stem cells (MSCs) are among the most important components of the osteosarcoma microenvironment and are reported to promote tumor progression. However, the means by which osteosarcoma cells modulate MSC behavior remains unclear. The aim of this study was to determine the effects of osteosarcoma cells on both the production of pro-tumor cytokines by mesenchymal stem cells (MSCs) and the osteogenic differentiation of MSCs. High level of transforming growth factor-ß (TGF-ß) was detected in three osteosarcoma cell lines. Conditioned media (CM) from the osteosarcoma cell lines Saos-2 and U2-OS were used to stimulate the cultured MSCs. We found that osteosarcoma cells promoted the production of IL-6 and VEGF in MSCs by inhibiting their osteogenic differentiation. Furthermore, TGF-ß in tumor CM was proved to be an important factor. The TGF-ß neutralizing antibody antagonized the effects induced by osteosarcoma CM. The inhibition of Smad2/3 by siRNA significantly decreased the production of IL-6 and VEGF in MSCs and induced their osteogenic differentiation. We also found that Smad2/3 enhanced the expression of ß-catenin in MSCs by decreasing the level of Dickkopf-1 (DKK1). Although the inhibition of ß-catenin did not affect the production of IL-6 or VEGF, or the gene expression of the early osteogenic markers Runx2 and ALP, it did enhance the gene expression of osteocalcin. Taken together, our data indicate that osteosarcoma cells secrete TGF-ß to maintain the stemness of MSCs and promote the production of pro-tumor cytokines by these cells.

[Application of enriched bone marrow compound with fibrin glue in repairing old radial bone defect in rabbits].

OBJECTIVE: To explore the feasibility of using enriched bone marrow (BM) compound with fibrin glue (FG) in repairing old radial bone defect. METHODS: Totally 36 New Zealand rabbits were equally randomized into three groups: simple FG group, BM+FG group, and enriched BM+FG group. A 1.5-cm segmental bone defect was made at the left radial in each animal. After one month, the defect was implanted with the engineered bone. Before implantation, a compound of enriched BM with FG underwent electron microscopy, long-term culture, and bacteriological culture. Four, 8, and 12 weeks after operations, the osteogenetic effect was evaluated using X-ray observation, HE staining, or Van Gieson staining, and a semi-quantitative analysis was performed. RESULTS: Electron microscopy showed enriched BM were compatible well with FG. No bacterial contamination or oncogenicity was observed after long-term culture. X-ray showed the repair effectiveness was significantly higher in BM+FG group and enriched BM+FG group than in simple FG group. Eight and 12 weeks after surgery, the Yang scores were significantly higher in enriched BM+FG group than in BM+FG group [(9.348±0.364évs.(7.984±0.229éìF=40.167ìP=0.001; (12.664±0.388)vs. (10.584±0.836é, F=20.3647ìP=0.004]. In addition, the Yang's scores at bone defects in BM+FG group and enriched BM+FG group were higher at the 12(th) week than in the 8(th) week. (F=36.004ìP=0.001; F=155.141ìP=0.000; respectively)The bone defects were repaired at varied degrees were histologically observed in BM+FG group and enriched BM+FG group during the observations. CONCLUSION: Implantation of BM+FG or enriched BM+FG are effective in repairing old radial bone defects, while simple FG shows not such effect.

Quaternized chitosan inhibits icaA transcription and biofilm formation by Staphylococcus on a titanium surface.

Our previous study (Z. X. Peng et al., Carbohydr. Polym. 81:275-283, 2010) demonstrated that water-soluble quaternary ammonium salts, which are produced by the reaction of chitosan with glycidyl trimethylammonium chloride, provide chitosan derivatives with enhanced antibacterial ability. Because biofilm formation is believed to comprise the key step in the development of orthopedic implant-related infections, we further evaluated the efficacy of hydroxypropyltrimethyl ammonium chloride chitosan (HACC) with different degrees of substitution (DS; referred to as HACC 6%, 18%, and 44%) in preventing biofilm formation on a titanium surface. We used a tissue culture plate method to quantify the biomass of Staphylococcus epidermidis and Staphylococcus aureus biofilms and found that HACC, especially HACC 18% and 44%, significantly inhibited biofilm formation compared to the untreated control, even at concentrations far below their MICs (P < 0.05). Scanning electron microscopy showed that inhibition of biofilm formation on titanium increased dramatically with increased DS and HACC concentrations. Confocal laser scanning microscopy indicated that growth of a preexisting biofilm on titanium was inhibited by concentrations of HACC 18% and 44% below their minimum biofilm eradication concentrations. We also demonstrated that HACC inhibited the expression of icaA, which mediates the production of extracellular polysaccharides, both in new biofilms and in preexisting biofilms on titanium. Our results indicate that HACC may serve as a new antibacterial agent to inhibit biofilm formation and prevent orthopedic implant-related infections.