An hydrophobically modified arginine peptide vector system effectively delivers DNA into human mesenchymal stem cells and maintains transgene expression with differentiation.

BACKGROUND: The ability of human mesenchymal stem cells (hMSCs) to differentiate into specific cells holds promise for therapeutic use in cell- or gene-based therapy. Genetic modification of hMSCs by introduction of therapeutic genes is an important tool for successful cell-mediated gene therapy. Similar to most primary cells, hMSCs are difficult to transfect with currently available gene delivery methods. Viral vectors are the most efficient DNA delivery system in stem cells. However, the use of viral vectors has disadvantages involving safety. To overcome these problems, nonviral gene delivery has been studied as an alternative strategy, and the cationic peptide was an efficient vector for transfecting various mammalian cell types. However, little is known about the capacity of this delivery method to transfect to hMSCs. In the present study, we examined the use of a short arginine peptide as a carrier for DNA delivery in hMSCs. METHODS: Plasmids containing the enhanced green fluorescent protein (EGFP) were transfected into hMSCs by arginine peptide in vitro. pEGFP delivery and EGFP expression were assessed by the fluorescence-activated cell sorting technique. RESULTS: The hydrophobically modified arginine peptide, palmitic acid-R15 (PA-R15), formed a condensed complex with DNA and successfully delivered the gene into hMSCs without compromising cell survival rate. Complexes readily internalized into hMSCs after a 4 h incubation, and PA-R15 showed higher cellular uptake compared to the unmodified arginine peptide (R15). In addition, transfected cells retained their stem cell properties, including the ability to differentiate into adipogenic and osteogenic lineages. Moreover, the erythropoietin (EPO) gene was successfully transfected into hMSCs and the cells produced EPO for 4 weeks. CONCLUSIONS: Hydrophobically modified arginine peptides are promising candidates with low toxicity. They comprise efficient nonviral gene delivery vectors for hMSCs and these may be used as a potential tool for basic research and the therapeutic application of hMSCs.

Immunomodulatory Effects of Monascus spp.-Fermented Sacccharina japonica Extracts on the Cytokine Gene Expression of THP-1 Cells.

The immunomodulatory effects of Monascus-fermented Saccharina japonica extract on anti- and pro-inflammatory cytokines gene expression of THP-1 cells were evaluated. Extracts of fermented samples showed higher phenolic, flavonoid, protein, and reducing sugar contents than unfermented one. Fermented samples were rich in many bioactive compounds determined by GC-MS analyses and showed cell viability greater than 85% in MTS assay. Regarding the anti-inflammatory and pro-inflammatory activities of the different samples, Q-PCR analyses revealed that IL-10 gene expression in THP-1 cells was significantly higher (p < 0.05) in cells treated with the SjMp or SjMk sample than those treated with the unfermented sample. Cells treated with the SjMp extract or lipopolysaccharide (LPS) showed significantly (p < 0.05) higher relative gene expression of IL-4 cytokine than cells treated with SjMk or SjU extracts. The relative gene expression of IFN-α was higher in cells treated with SjMp followed by LPS, SjMk, and SjU. TGF-ß expression was higher in LPS-stimulated cells followed by SjMk and other samples. Cells treated with SjMp exhibited significantly higher pro-inflammatory (IL-6, IL-8, TNF-α, and NF-κB) cytokine gene expression than cells treated with SjU. These results revealed that extracts from S. japonica fermented with Monascus spp. regulate cytokine gene expression. Graphical abstract ᅟ.

N-terminal domain of the beta-propeller phytase of Pseudomonas sp. FB15 plays a role for retention of low-temperature activity and catalytic efficiency.

Pseudomonas sp. FB15 producing phytase with a high activity at low temperature was isolated from foreshore soil. The phytase gene was cloned and sequenced. Molecular modeling predictions using its amino acid sequence indicated that it belongs to the group of beta-propeller phytases (BPPs). However, it differs from the conventional structure of BPPs in the number of propeller-shaped domains. Most BPPs have a propeller with six blades that consist of four or five beta-sheets. However, the BPP from the isolated strain contained a C-terminal domain containing a conserved BPP sequence and also an N-terminal domain consisting of an additional 275 amino acids. The characterization of recombinant BPP from Pseudomonas sp. FB15 (PSphy) revealed the highest activity to be at pH 6 and 40 °C. Additionally, it showed that more than 80% of optimal activity was retained at a relatively low temperature of 25 °C. In addition, CaCl2 was required for activity, and the optimal concentration of CaCl2 was 4 mM. The activity at low temperature was reduced in a recombinant protein from which the N-terminal domain was removed, as was catalytic efficiency. These results suggest that the N-terminal domain is required for the low temperature activity and high catalytic efficiency of PSphy.

Simultaneous blockade of VEGF and Dll4 by HD105, a bispecific antibody, inhibits tumor progression and angiogenesis.

Several angiogenesis inhibitors targeting the vascular endothelial growth factor (VEGF) signaling pathway have been approved for cancer treatment. However, VEGF inhibitors alone were shown to promote tumor invasion and metastasis by increasing intratumoral hypoxia in some preclinical and clinical studies. Emerging reports suggest that Delta-like ligand 4 (Dll4) is a promising target of angiogenesis inhibition to augment the effects of VEGF inhibitors. To evaluate the effects of simultaneous blockade against VEGF and Dll4, we developed a bispecific antibody, HD105, targeting VEGF and Dll4. The HD105 bispecific antibody, which is composed of an anti-VEGF antibody (bevacizumab-similar) backbone C-terminally linked with a Dll4-targeting single-chain variable fragment, showed potent binding affinities against VEGF (KD: 1.3 nM) and Dll4 (KD: 30 nM). In addition, the HD105 bispecific antibody competitively inhibited the binding of ligands to their receptors, i.e., VEGF to VEGFR2 (EC50: 2.84 ± 0.41 nM) and Dll4 to Notch1 (EC50: 1.14 ± 0.06 nM). Using in vitro cell-based assays, we found that HD105 effectively blocked both the VEGF/VEGFR2 and Dll4/Notch1 signaling pathways in endothelial cells, resulting in a conspicuous inhibition of endothelial cell proliferation and sprouting. HD105 also suppressed Dll4-induced Notch1-dependent activation of the luciferase gene. In vivo xenograft studies demonstrated that HD105 more efficiently inhibited the tumor progression of human A549 lung and SCH gastric cancers than an anti-VEGF antibody or anti-Dll4 antibody alone. In conclusion, HD105 may be a novel therapeutic bispecific antibody for cancer treatment.

RNA interference in vitro and in vivo using an arginine peptide/siRNA complex system.

Efficient delivery systems are required to exploit the enormous potential of RNA interference. We introduced an arginine peptide-based small-interference RNA (siRNA) delivery system for in vitro and in vivo RNA interference. Arginine peptides formed stable complexes with siRNA and transduced siRNA into COS-7 cells in vitro, resulting in efficient gene silencing. The intracellular path of the peptide/siRNA complex was investigated in live cells using fluorescent labeling and confocal microscopy. At 24h after transfection, most of the siRNA signals were observed in the perinuclear region, indicating that siRNA was targeted to the perinuclear region for interactions with RNA-induced silencing complex (RISC). Effective in vivo RNA interference was achieved in a mouse model bearing a subcutaneous tumor. Intratumoral administration of HER-2-specific siRNA/peptide complexes resulted in a marked reduction of tumor growth. Body weight monitoring during treatment showed that our delivery system was nontoxic. Our approach offers the potential for siRNA delivery in various in vitro and in vivo applications.