Trehalose Maintains Bioactivity and Promotes Sustained Release of BMP-2 from Lyophilized CDHA Scaffolds for Enhanced Osteogenesis In Vitro and In Vivo

Calcium phosphate (Ca-P) scaffolds have been widely employed as a supportive matrix and delivery system for bone tissue engineering. Previous studies using osteoinductive growth factors loaded Ca-P scaffolds via passive adsorption often experience issues associated with easy inactivation and uncontrolled release. In present study, a new delivery system was fabricated using bone morphogenetic protein-2 (BMP-2) loaded calcium-deficient hydroxyapatite (CDHA) scaffold by lyophilization with addition of trehalose. The in vitro osteogenesis effects of this formulation were compared with lyophilized BMP-2/CDHA construct without trehalose and absorbed BMP-2/CDHA constructs with or without trehalose. The release characteristics and alkaline phosphatase (ALP) activity analyses showed that addition of trehalose could sufficiently protect BMP-2 bioactivity during lyophilization and achieve sustained BMP-2 release from lyophilized CDHA construct in vitro and in vivo. However, absorbed BMP-2/CDHA constructs with or without trehalose showed similar BMP-2 bioactivity and presented a burst release. Quantitative real-time PCR (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) demonstrated that lyophilized BMP-2/CDHA construct with trehalose (lyo-tre-BMP-2) promoted osteogenic differentiation of bone marrow stromal cells (bMSCs) significantly and this formulation could preserve over 70% protein bioactivity after 5 weeks storage at 25°C. Micro-computed tomography, histological and fluorescent labeling analyses further demonstrated that lyo-tre-BMP-2 formulation combined with bMSCs led to the most percentage of new bone volume (38.79% ±5.32%) and area (40.71% ±7.14%) as well as the most percentage of fluorochrome stained bone area (alizarin red S: 2.64% ±0.44%, calcein: 6.08% ±1.37%) and mineral apposition rate (4.13±0.62 µm/day) in critical-sized rat cranial defects healing. Biomechanical tests also indicated the maximum stiffness (118.17±15.02 Mpa) and load of fracture (144.67±16.13 N). These results lay a potential framework for future study by using trehalose to preserve growth factor bioactivity and optimize release profile of Ca-P based delivery system for enhanced bone regeneration.     

Bicyclic α,ω-dicarboxylic acid derivatives from a colonial tunicate of the family Polyclinidae

In the course of our search for bioactive metabolites from a colonial tunicate of the family Polyclinidae, six new (1–6) cyclic fatty acid derivatives were isolated. Their planar structures were established on the basis of NMR and MS spectroscopic analyses. The relative configuration was determined by NOESY experiment. Compounds 1–6 represent a fused bicyclic skeleton possibly derived from α,ω-dicarboxylic acids such as eicosanedioic acid or docosanedioic acid via a Diels–Alder type of cyclization. Compounds 1–4 and 6 showed mild cytotoxicity against a panel of five human solid tumor cell lines.     

Two-component sensor RhpS promotes induction of Pseudomonas syringae type III secretion system by repressing negative regulator RhpR

The Pseudomonas syringae type III secretion system (T3SS) is induced during interaction with the plant or culture in minimal medium (MM). How the bacterium senses these environments to activate the T3SS is poorly understood. Here, we report the identification of a novel two-component system (TCS), RhpRS, that regulates the induction of P. syringae T3SS genes. The rhpR and rhpS genes are organized in an operon with rhpR encoding a putative TCS response regulator and rhpS encoding a putative biphasic sensor kinase. Transposon insertion in rhpS severely reduced the induction of P. syringae T3SS genes in the plant as well as in MM and significantly compromised the pathogenicity on host plants and hypersensitive response-inducing activity on nonhost plants. However, deletion of the rhpRS locus allowed the induction of T3SS genes to the same level as in the wild-type strain and the recovery of pathogenicity upon infiltration into plants. Overexpression of RhpR in the deltarhpRS deletion strain abolished the induction of T3SS genes. However, overexpression of RhpR in the wild-type strain or overexpression of RhpR(D70A), a mutant of the predicted phosphorylation site of RhpR, in the deltarhpRS deletion strain only slightly reduced the induction of T3SS genes. Based on these results, we propose that the phosphorylated RhpR represses the induction of T3SS genes and that RhpS reverses phosphorylation of RhpR under the T3SS-inducing conditions. Epistasis analysis indicated that rhpS and rhpR act upstream of hrpR to regulate T3SS genes.     

Genetic diversity and genetic structure of different populations of the endangered species Davidia involucrata in China detected by inter-simple sequence repeat analysis

Dove tree (Davidia involucrate) is a Tertiary relic species endemic to China and is reputed to be a ‘living fossil’ in the plant kingdom. Genetic diversity and genetic structure of this species were analyzed for its conservation and management, using inter-simple sequence repeat (ISSR) data obtained from eight populations distributed throughout seven provinces of China. A relatively high level of genetic diversity, at both population and species levels, was detected using the POPGENE software. Analysis of molecular variance (AMOVA) revealed a moderate level of among-population variation (i.e., 33.21%). The genetic structure of dove tree was closely consistent with their isolated topographical distribution region based on the results of the STRUCTURE, POPGENE-UPGMA and PCA analysis. It is postulated that the relatively high level of genetic diversity has been maintained because of: (a) the original wild geographical distribution, (b) propagation through outcrossing seeds or root suckers, (c) the longevity of individuals and (d) the relatively little human disturbance. Genetic drift and restricted gene are important factors affecting genetic differentiation. There was no significant correlation between geographical distances and a pairwise comparison with genetic distances, as analyzed by the Mantel test, but the clustering result of genetic diversity was consistent with their isolated topographical distribution regions. Thus, maintaining the stable special habitats associated with this species is recommended for the in situ conservation. Furthermore, it is important to develop an effective seed germination system for the maintenance of an ex situ conservation pool of the germplasm resources.