No abstract

Protein lysine succinylation, an emerging protein post-translational modification widespread among eukaryotic and prokaryotic cells, represents an important regulator of cellular processes. However, the extent and function of lysine succinylation in Mycobacterium tuberculosis, especially extensively drug-resistant strain, remain elusive. Combining protein/peptide prefractionation, immunoaffinity enrichment, and LC-MS/MS analysis, a total of 686 succinylated proteins and 1739 succinylation sites of M. tuberculosis were identified, representing the first global profiling of M. tuberculosis lysine succinylation. The identified succinylated proteins are involved in a variety of cellular functions such as metabolic processes, transcription, translation, and stress responses and exhibit different subcellular localization via GO, protein interaction network, and other bioinformatic analysis. Notably, proteins involved in protein biosynthesis and carbon metabolism are preferred targets of lysine succinylation. Moreover, two prevalent sequence patterns: EK(suc) and K*****K(suc), can be found around the succinylation sites. There are 109 lysine-succinylated homologues in E. coli, suggesting highly conserved succinylated proteins. Succinylation was found to occur at the active sites predicted by Prosite signature including Rv0946c, indicating that lysine succinylation may affect their activities. There is extensive overlapping between acetylation sites and succinylation sites in M. tuberculosis. Many M. tuberculosis metabolic enzymes and antibiotic resistance proteins were succinylated. This study provides a basis for further characterization of the pathophysiological role of lysine succinylation in M. tuberculosis.

Strategies to control neovascularization in damaged tissues remain a key issue in regenerative medicine. Unlike most reported desferrioxamine (DFO)-loaded systems where DFO demonstrates a burst release, here we attain zero-order release behavior above 40 days. This outcome was achieved by blending DFO with silk nanofibers with special hydrophilic–hydrophobic properties. The special silk nanofibers showed strong physical binding capacity with DFO, avoiding chemical cross-linking. Using these new biomaterials in vivo in a rat wound model suggested that the DFO-loaded silk nanofiber hydrogel systems stimulated angiogenesis by the sustained release of DFO, but also facilitated cell migration and tissue ingrowth. These features resulted in faster formation of a blood vessel network in the wounds, as well improved healing when compared to the free DFO system. The DFO-loaded systems are also suitable for the regeneration of other tissues, such as nerve and bone, suggesting universality in the biomedical field.

The redevelopment/regeneration pattern of amputated limbs from a blastema in salamander suggests that enhanced regeneration might be achieved by mimicking the developmental microenvironment. Inspired by the discovery that the expression of magnesium transporter‐1 (MagT1), a selective magnesium (Mg) transporter, is significantly upregulated in the endochondral ossification region of mouse embryos, a Mg‐enriched 3D culture system is proposed to provide an embryonic‐like environment for stem cells. First, the optimum concentration of Mg ions (Mg 2+ ) for creating the osteogenic microenvironment is screened by evaluating MagT1 expression levels, which correspond to the osteogenic differentiation capacity of stem cells. The results reveal that Mg 2+ selectively activates the mitogen‐activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway to stimulate osteogenic differentiation, and Mg 2+ influx via MagT1 is profoundly involved in this process. Then, Mg‐enriched microspheres are fabricated at the appropriate size to ensure the viability of the encapsulated cells. A series of experiments show that the Mg‐enriched microenvironment not only stimulates the osteogenic differentiation of stem cells but also promotes neovascularization. Obvious vascularized bone regeneration is achieved in vivo using these Mg‐enriched cell delivery vehicles. The findings suggest that biomaterials mimicking the developmental microenvironment might be promising tools to enhance tissue regeneration.

No abstract

Small cell lung cancer (SCLC) is a rapidly progressing, incurable cancer that frequently spreads to bone. New insights are needed to identify therapeutic targets to prevent or retard SCLC metastatic progression. Human SCLC SBC-5 cells in mouse xenograft models home to skeletal and nonskeletal sites, whereas human SCLC SBC-3 cells only pervade nonskeletal sites. Because microRNAs (miRNA) often act as tumor regulators, we investigated their role in preclinical models of SCLC. miRNA expression profiling revealed selective and reduced expression of miRNA (miR)-335 and miR-29a in SBC-5 cells, compared with SBC-3 cells. In SBC-5 cells, miR-335 expression correlated with bone osteolytic lesions, whereas miR-29a expression did not. Overexpression of miR-335 in SBC-5 cells significantly reduced cell migration, invasion, proliferation, colony formation, and osteoclast induction in vitro. Importantly, in miR-335 overexpressing SBC-5 cell xenografts (n ¼ 10), there were minimal osteolytic lesions in the majority of mice and none in three mice. Expression of RANK ligand (RANKL) and insulin-like growth factor-I receptor (IGF-IR), key mediators of bone metastases, were elevated in SBC-5 as compared with SBC-3 cells. Mechanistically, overexpression of miR-335 in SBC-5 cells reduced RANKL and IGF-IR expression. In conclusion, loss of miR-335 promoted SCLC metastatic skeletal lesions via deregulation of IGF-IR and RANKL pathways and was associated with metastatic osteolytic skeletal lesions.

Osteochondral repair remains a major challenge in current clinical practice despite significant advances in tissue engineering. In particular, the lateral integration of neocartilage into surrounding native cartilage is a difficult and inadequately addressed problem that determines the success of tissue repair. Here, a novel design of an integral bilayer scaffold combined with a photocurable silk sealant for osteochondral repair is reported. First, we fabricated a bilayer silk scaffold with a cartilage layer resembling native cartilage in surface morphology and mechanical strength and a BMP-2-loaded porous subchondral bone layer that facilitated the osteogenic differentiation of BMSCs. Second, a TGF-β3-loaded methacrylated silk fibroin sealant (Sil-MA) exhibiting biocompatibility and good adhesive properties was developed and confirmed to promote chondrocyte migration and differentiation. Importantly, this TGF-β3-loaded Sil-MA hydrogel provided a bridge between the cartilage layer of the scaffold and the surrounding cartilage and then guided new cartilage to grow towards and replace the degraded cartilage layer from the surrounding native cartilage in the early stage of knee repair. Thus, osteochondral regeneration and superior lateral integration were achieved in vivo by using this composite. These results demonstrate that the new approach of marginal sealing around the cartilage layer of bilayer scaffolds with Sil-MA hydrogel has tremendous potential for clinical use in osteochondral regeneration.

MicroRNAs (miRNAs) have been identified to play important functions during osteoblast proliferation, differentiation, and apoptosis. The miR-17~92 cluster is highly conserved in all vertebrates. Loss-of-function of the miR-17-92 cluster results in smaller embryos and immediate postnatal death of all animals. Germline hemizygous deletions of MIR17HG are accounted for microcephaly, short stature, and digital abnormalities in a few cases of Feingold syndrome. These reports indicate that miR-17~92 may play important function in skeletal development and mature. To determine the functional roles of miR-17~92 in bone metabolism as well as osteoblast proliferation and differentiation. Murine embryonic stem cells D3 and osteoprogenitor cell line MC3T3-E1 were induced to differentiate into osteoblasts; the expression of miR-17-92 was assayed by quantitative real-time RT-PCR. The skeletal phenotypes were assayed in mice heterozygous for miR-17~92 (miR-17~92 (+/Δ) ). To determine the possibly direct function of miR-17~92 in bone cells, osteoblasts from miR-17~92 (+/Δ) mice were investigated by ex vivo cell culture. miR-17, miR-92a, and miR-20a within miR-17-92 cluster were expressed at high level in bone tissue and osteoblasts. The expression of miR-17-92 was down-regulated along with osteoblast differentiation, the lowest level was found in mature osteoblasts. Compared to wildtype controls, miR-17-92 (+/Δ) mice showed significantly lower trabecular and cortical bone mineral density, bone volume and trabecular number at 10 weeks old. mRNA expression of Runx2 and type I collagen was significantly lower in bone from miR-17-92 (+/Δ) mice. Osteoblasts from miR-17-92 (+/Δ) mice showed lower proliferation rate, ALP activity and less calcification. Our research suggests that the miR-17-92 cluster critically regulates bone metabolism, and this regulation is mostly through its function in osteoblasts.