Guided bone regeneration (GBR) technique is widely used in the treatment of bone defects caused by peri-implantitis, periodontal disease, etc. However, the GBR membranes commonly used in clinical treatments currently have no antibacterial activity. Therefore, in this study, sequential layer-by-layer electrospinning and electrospraying techniques were utilized to prepare a gelatin (Gln) and chitosan (CS) composite GBR membrane containing hydroxyapatite nanoparticles (nHAp) and antimicrobial peptide (Pac-525)-loaded PLGA microspheres (AMP@PLGA-MS), which was supposed to have osteogenic and antibacterial activities. The scanning electron microscope (SEM) observation showed that the morphology of the nanofibers and microspheres could be successfully produced. The diameters of the electrospun fibers with and without nHAp were 359 ± 174 nm and 409 ± 197 nm, respectively, and the mechanical properties of the membrane were measured according to the tensile stress-strain curve. Both the involvement of nHAp and the chemical crosslinking were able to enhance their tensile strength. In vitro cell culture of rat bone marrow mesenchymal stem cells (rBMSCs) indicated that the Gln/CS composite membrane had an ideal biocompatibility with good cell adhesion, spreading, and proliferation. In addition, the Gln/CS membrane containing nHAp could promote osteogenic differentiation of rBMSCs. Furthermore, according to the in vitro drug release assay and antibacterial experiments, the composite GBR membrane containing AMP@PLGA-MS exhibited a long-term sustained release of Pac-525, which had bactericidal activity within one week and antibacterial activity for up to one month against two kinds of bacteria, S. aureus and E. coli. Our results suggest that the antimicrobial peptide-loaded Gln/CS composite membrane (AMP@PLGA-MS@Gln/CS/nHAp) has a great promise in bone generation-related applications for the unique functions of guiding bone regeneration and inhibiting bacterial infection as well.

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is regulated by many factors and signal pathways, including transcription factors such as Sox9 and microRNAs. MiR‐335‐5p has been previously reported to regulate osteogenic and adipogenic differentiations of MSCs, but its role in chondrogenic differentiation of MSC remains unknown. In this study, we found that miR‐335‐5p and its host gene Mest are co‐expressed and greatly upregulated during mouse MSCs (mMSCs) chondrogenesis. Overexpression of miR‐335‐5p in mMSCs increased expression of chondrogenic marker genes. Molecular mechanism explorations revealed that miR‐335‐5p targets Daam1 and ROCK1, a set of negative regulators of Sox9; Sox9 downregulates the expression of miR‐29a and 29b, both negative regulators of Mest expression, thus forming a positive loop from miR‐335‐5p to Sox9 to Mest/miR‐335‐5p. In addition, miR‐335‐5p targets DKK1 during mMSC chondrogenic differentiation to increase β‐catenin/TCF activity, which leads to increased level of Mest transcription. These data showed miR‐335‐5p positively regulates MSC chondrogenesis, and two positive feedback loops are identified for the expression of miR‐335‐5p and its host gene Mest during the early phase of mMSC chondrogenic differentiation. © 2014 American Society for Bone and Mineral Research.

Various methods for editing specific sites in the Escherichia coli chromosome are available, and gene-size (∼1 kb) integration into a single site or to introduce deletions, short insertions or point mutations into multiple sites can be conducted in a short period of time. However, a method for rapidly integrating multiple gene-size sequences into different sites has not been developed yet. Here, we describe a method and plasmid system that makes it possible to simultaneously insert genes into multiple specific loci of the E. coli genome without the need for chromosomal markers. The method uses a CRISPR-Cas12a system to eliminate unmodified cells by double-stranded DNA cleavage in conjunction with the phage-derived λ-Red recombinases to facilitate recombination between the chromosome and the donor DNA. We achieved the insertion of up to 3 heterologous genes in one round of recombination and selection. To demonstrate the practical application of this gene-insertion method, we constructed a recombinant E. coli producing an industrially useful chemical, 5-aminolevulinic acid (ALA), with high-yield. Moreover, a similar two-plasmid system was built to edit the genome of the extremophile Halomonas bluephagenesis.

The endothelial-to-mesenchymal transition (EndMT) is an important source of fibrotic cells in idiopathic pulmonary fibrosis (IPF). However, how endothelial cells (ECs) are activated and how EndMT impact IPF remain largely elusive. Here, we use unsupervised pseudotemporal analysis to recognize the heterogeneity of ECs and reconstruct EndMT trajectory of bleomycin (BLM)-treated Tie2^creER/+;Rosa26^tdTomato/+ IPF mice. Genes like C3ar1 and Lgals3 (protein name galectin-3) are highly correlated with the transitional pseudotime, whose expression is gradually upregulated during the fate switch of ECs from quiescence to activation in fibrosis. Inhibition of galectin-3 via siRNA or protein antagonists in mice could alleviate the pathogenesis of IPF and the transition of ECs. With the stimulation of human pulmonary microvascular endothelial cells (HPMECs) by recombinant proteins and/or siRNAs for galectin-3 in vitro, β-catenin/GSK3β signaling and its upstream regulator AKT are perturbed, which indicates they mediate the EndMT progress. These results suggest that EndMT is essential to IPF process and provide potential therapeutic targets for vascular remodeling.