Controlled release of minocycline in hydroxyapatite/chitosan composite for periodontal bone defect repair.

The aim was to obtain bone repair materials with sustained release of minocycline and evaluate the effect in periodontal bone defect repair. Two complex material, hydroxyapatite/chitosan (HA/CS) and minocycline-hydroxyapatite/chitosan (Mino-HA/CS), were prepared by the co-precipitation method. The physical and chemical property, cytotoxicity, release of minocycline and the bacteriostasis examination of the materials were evaluated, they were applied to the rabbit model of mandible bone defect to evaluate their effects on the regeneration of periodontal bone defect. After minocycline was added to HA/CS, the setting time of the material was prolonged, the compressive strength was reduced and the pore size and porosity were increased significantly. The pH value did not change obviously and stayed in the neutral range. Mino-HA/CS could promote the growth of osteoblasts effectively compared with control medium. In vivo, Mino-HA/CS material showed better effect of promoting periodontal bone formation.

LAMP2: a major update of the database linking antimicrobial peptides.

Antimicrobial peptides (AMPs) have been regarded as a potential weapon to fight against drug-resistant bacteria, which is threating the globe. Thus, more and more AMPs had been designed or identified. There is a need to integrate them into a platform for researchers to facilitate investigation and analyze existing AMPs. The AMP database has become an important tool for the discovery and transformation of AMPs as agents. A database linking antimicrobial peptides (LAMPs), launched in 2013, serves as a comprehensive tool to supply exhaustive information of AMP on a single platform. LAMP2, an updated version of LAMP, holds 23 253 unique AMP sequences and expands to link 16 public AMP databases. In the current version, there are more than 50% (12 236) sequences only linking a single database and more than 45% of AMPs linking two or more database links. Additionally, updated categories based on primary structure, collection, composition, source and function have been integrated into LAMP2. Peptides in LAMP2 have been integrated in 8 major functional classes and 38 functional activities. More than 89% (20 909) of the peptides are experimentally validated peptides. A total of 1924 references were extracted and regarded as the evidence for supporting AMP activity and cytotoxicity. The updated version will be helpful to the scientific community.

Mir-1287 suppresses the proliferation, invasion, and migration in hepatocellular carcinoma by targeting PIK3R3.

Mature microRNAs (miRNAs) are a class of small noncoding RNA molecules involved in regulation of post-translational gene expression. Although aberrant levels of miRNAs have been found in various tumor tissues, their importance in tumor development and the molecular basis of their regulatory role remain unclear. Our bioinformatic analysis on The Cancer Genome Atlas database and microarray-based comparison of miRNA in different cell lines revealed that the level of mir-1287 is suppressed in hepatocellular carcinoma (HCC) cells. When upregulated, mir-1287 can reduce the tumorigenesis phenotypes of HCC cells in several in vitro models. We further found that mir-1287 directly targets messenger RNA encoding PIK3R3, which is a tumor-promoting factor acting in several pathways linked to tumorigenesis. Our study suggests that aberrant suppression of mir-1287 is potentially responsible for the development of HCC, and miRNA-based strategies may be developed for efficient detection and treatment of HCC.

MicroRNA-493 suppresses hepatocellular carcinoma tumorigenesis through down-regulation of anthrax toxin receptor 1 (ANTXR1) and R-Spondin 2 (RSPO2).

Hepatocellular carcinoma (HCC) is known as a highly prevalent cancer with a poor prognosis and short survival time, despite intensive research and clinical efforts. Increasing numbers of studies have reported that microRNAs are involved in the malignant behavior of hepatocellular carcinoma cells via directly targeting multiple oncogenes or tumor suppressors. Here, we report that the expression of microRNA-493 (miR-493) is decreased in HCC cell lines and in tumor tissues. Overexpression of miR-493 in HCC cells dramatically inhibited cell proliferation and colony-formation in vitro and inhibited tumor formation of HCC cell xenografts in vivo. miR-493 also suppressed cell migration and invasion in HCC cell lines. Novel targets ANTXR1 and RSPO2 were confirmed to be suppressed by miR-493 directly, and overexpression of ANTXR1 and RSPO2 could restore tumorigenesis in miR-493 treated HCC cell. Moreover, Wnt/ß-catenin signaling pathway, which was reported to be activated by ANTXR1 and RSPO2, was also inhibited by miR-493 overexpression and might be involved in anti-tumor function of miR-493. These findings suggest that miR-493 acts as a negative regulator in hepatocellular carcinoma progression and may be a potential therapeutic target for HCC.

Serine protease inhibitor kazal-type 6 inhibits tumorigenesis of human hepatocellular carcinoma cells via its extracellular action.

Hepatocellular carcinoma (HCC) causes significant medical burdens worldwide. Diagnosis, especially in the early stages, is still challenging. Therapeutic options are limited and often ineffective. Although several risk factors have been known important for development of HCC, the molecular basis of the process is rather complex and has not been fully understood. We have found that a subpopulation of HCC cells which are resistant to oncolytic parvovirus H1 superinfection highly express serine protease inhibitor Kazal-type 6 (SPINK6). This protein is specifically reduced in all HCC cell lines and tissues we analyzed. When upregulated, SPINK6 could suppress the malignant phenotypes of the HCC cells in several in vitro models. The putative tumor suppression role of SPINK6 is, however, independent of its protease inhibitory activity. To suppress the malignancy of HCC cells, SPINK6 has to be secreted to trigger signals which regulate an intracellular signaling molecule, ERK1/2, as well as a series of downstream factors involved in cell cycle progression, apoptosis and migration. Our study supports that SPINK6 is an important tumor suppressor in liver, and further investigations may help develop more effective diagnostic and therapeutic approaches.

Controlled release by novel lysostaphin-loaded hydroxyapatite/chitosan composites.

Lysostaphin is highly effective on eliminating methicillin resistant Staphylococcus aureus (MRSA). In order to achieve controlled release of lysostaphin, a biocompatible drug carrier is needed. Hydroxyapatite/chitosan (HA/CS) composites were chosen to carry lysostaphin and sample composites with different weight ratios of HA to CS, including 80/20, 70/30, 60/40, and 40/60, were prepared. Multiple analyses were performed to determine the structural and physicochemical properties of the composites, including scanning electron microscopy, X-ray diffraction and Fourier transform infrared spectroscopy. We immersed HA/CS composites loaded with 1 wt% lysostaphin to test in vitro release activity and cultured MC3T3-E1 cells to carry out biocompatibility test. The result of the release behavior of the composites revealed that the controlled release of lysostaphin from 60/40 HA/CS composites was the highest release rate of (87.4 ± 2.8)%, which lasted for 120 hours. In biocompatibility testing, MC3T3-E1 cells were able to proliferate on the surface of these composites, and the extract liquid from the composites could increase the growth of the cells. These results demonstrate the controlled release of lysostaphin from HA/CS composites and their biocompatibility, suggesting the potential application of these composites to bone injury and infection applications.

CD226 protein is involved in immune synapse formation and triggers Natural Killer (NK) cell activation via its first extracellular domain.

CD226, an activating receptor that interacts with the ligands CD155 and CD112, activates natural killer (NK) cells via its immunoreceptor tyrosine-based activatory motif (ITAM). There are two extracellular domains of CD226; however, the comparative functional relevance of these domains remains unknown. In this study, two different deletion mutants, rCD226-ECD1 (the first extracellular domain) and rCD226-ECD (full extracellular domains), were recombinantly expressed. We observed that rCD226-ECD1, similar to rCD226-ECD, specifically bound to ligand-positive cell lines and that this interaction could be competitively blocked by an anti-CD226 mAb. In addition, rCD226-ECD1 was able to block the binding of CD112 mAb to tumor cells in a competitive binding assay. Importantly, based on surface plasmon resonance (SPR), we determined that rCD226-ECD1, similar to rCD226-ECD, directly bound to its ligand CD155 on a protein chip. Functionally, NK cell cytotoxicity against K562 or HeLa cells was blocked by rCD226-ECD1 by reducing the expression of CD69 and granzyme B, indicating the critical role of ECD1 in NK cell activation. We also examined the role of rCD226-ECD1 in effector/target interactions by using rCD226-ECD to block these interactions. Using flow cytometry, we found that the number of conjugates between IL-2-dependent NKL cells and HeLa cells was reduced and observed that the formation of immune synapses was also decreased under confocal microscopy. In addition, we prepared two anti-rCD226-ECD1 agonistic antibodies, 2E6 and 3B9. Both 2E6 and 3B9 antibodies could induce the phosphorylation of ERK in NK-92 cells. Taken together, our results show that CD226 functions via its first extracellular domain.

Expression, purification and characterization of recombinant human serine proteinase inhibitor Kazal-type 6 (SPINK6) in Pichia pastoris.

Human serine proteinase inhibitor Kazal-type 6 (SPINK6) belongs to the medically important SPINK family. Malfunctions of SPINK members are linked to many diseases, including pancreatitis, skin barrier defects, and cancer. SPINK6 has been shown to selectively inhibit Kallikrein-related peptidases (KLKs) in human skin. As a SPINK protein, it contains a typical Kazal domain, which requires three intramolecular disulfide bonds for correct folding and activity. Preparation of functional protein is a prerequisite for studying this important human factor. Here, we report the successful generation of tagless SPINK6 using a yeast expression system. The recombinant protein was secreted and purified by cation exchange and size-exclusion chromatography. The protein identity was confirmed by MALDI-TOF MS and N-terminal sequencing. Pichia pastoris-derived recombinant human SPINK6 (rhSPINK6) showed higher inhibitory activity against Kallikrein-related peptidase 14 (KLK14) (K(i)=0.16 nM) than previously reported Escherichia coli-derived rhSPINK6 (K(i)=0.5 nM). This protein also exhibited moderate inhibition of bovine trypsin (K(i)=33 nM), while previous E. coli-derived rhSPINK6 did not. The results indicate that P. pastoris is a better system to generate active rhSPINK6, warranting further studies on this medically important SPINK family candidate.

Rapid immunosorting of transmembrane proteins of Lymphocytes from a cDNA expression library of COS-1 cells.

The proteins on lymphocyte surface play important roles in a wide range of immunological processes, but the profile and characterization of surface proteins remain to be further investigated, among which the method for fast screening of surface proteins needs to be established. In this study, a conventional cDNA clone library of hepatic lymphocytes from C57BL/6 mouse was constructed, and then the cDNA was inserted into a recombinant expression vector pSecTag-attR with a signal peptide and tag protein for fluorescence screening. The recombinant cDNA expression library was transfected into COS-1 cells, and the transfected cells with the expressed membrane proteins were labeled by fluorescence antibodies and isolated by fluorescence activated cell sorting. After two cycles of sorting, the purity of fluorescence positive cells with membrane proteins was up to 98%, and the representative membrane molecules on lymphocytes such as CD3, CD4, CD8, NK1.1 and NKG2D were detected in the library. These results demonstrated that the cDNA expression library containing transmembrane proteins provided an efficient and fast tool for the study of transmembrane proteins on hepatic lymphocytes.