The Use of Chitosan Nanoparticles for Delivery of CpG ODN in Treatment of Allergic Balb/C Mice.

Background: This study aims to prepare high stability chitosan nanoparticles (CNP) and examine the ability of CNP in CpG-ODN delivery when treating allergic mice model. Methods: Preparation and characterization of CNP were performed by ionic gelation, dynamic light scattering, and zeta sizer. The CNP cytotoxicity and activation ability of CpG ODN delivered with CNP were tested using a cell counting kit-8 and Quanti blue method. Allergic mice were injected intraperitoneal with 10 ug ovalbumin on day 0 and 7, and then treated with intranasal CpG ODN/CpG ODN, delivered with CNP/CNP, on the third week three times per week for three weeks. The ELISA method measured cytokine and IgE profiles in the allergic mice's plasma and spleen. Results: CNP results have sizes 27.73 nm±3.67 dan 188.23 nm±53.47, spherical in shape and non-toxic, and did not alter the NF-κB activation of CpG ODN in RAW-blue cells. The application of CpG ODN delivered by chitosan nanoparticles shows no statistical difference between groups of IFN-γ, IL-10, and IL-13 in Balb/c mice's plasma and spleen, in contrast with IgE level. Conclusions: The results showed that using chitosan nanoparticles as a delivery system for CpG ODN has the potency to safely CpG ODN efficacy.

Enhancement of the Immunostimulatory Effect of Phosphodiester CpG Oligodeoxynucleotides by an Antiparallel Guanine-Quadruplex Structural Scaffold.

Guanine-quadruplex-based CpG oligodeoxynucleotides (G4 CpG ODNs) have been developed as potent immunostimulatory agents with reduced sensitivity to nucleases. We designed new monomeric G4 ODNs with an antiparallel topology using antiparallel type duplex/G4 ODNs as robust scaffolds, and we characterized their topology and effects on cytokine secretion. Based on circular dichroism analysis and quantification of mRNA levels of immunostimulatory cytokines, it was found that monomeric antiparallel G4 CpG ODNs containing two CpG motifs in the first functional loop, named G2.0.0, could maintain antiparallel topology and generate a high level of immunostimulatory cytokines in RAW264 mouse macrophage-like cell lines. We also found that the flanking sequence in the CpG motif altered the immunostimulatory effects. Gc2c.0.0 and Ga2c.0.0 are monomeric antiparallel G4 CpG ODNs with one cytosine in the 3' terminal and one cytosine/adenine in the 5' terminal of CpG motifs that maintained the same resistance to degradation in serum as G2.0.0 and improved interleukin-6 production in RAW264 and bone marrow-derived macrophages. The immunostimulatory activity of antiparallel G4 CpG ODNs is superior to that of linear natural CpG ODNs. These results provide insights for the rational design of highly potent CpG ODNs using antiparallel G4 as a robust scaffold.

Employment of 1-Methoxy-5-Ethyl Phenazinium Ethyl Sulfate as a Stable Electron Mediator in Flavin Oxidoreductases-Based Sensors.

In this paper, a novel electron mediator, 1-methoxy-5-ethyl phenazinium ethyl sulfate (mPES), was introduced as a versatile mediator for disposable enzyme sensor strips, employing representative flavin oxidoreductases, lactate oxidase (LOx), glucose dehydrogenase (GDH), and fructosyl peptide oxidase (FPOx). A disposable lactate enzyme sensor with oxygen insensitive Aerococcus viridans-derived engineered LOx (AvLOx), with A96L mutant as the enzyme, was constructed. The constructed lactate sensor exhibited a high sensitivity (0.73 ± 0.12 µA/mM) and wide linear range (0-50 mM lactate), showings that mPES functions as an effective mediator for AvLOx. Employing mPES as mediator allowed this amperometric lactate sensor to be operated at a relatively low potential of +0.2 V to 0 V vs. Ag/AgCl, thus avoiding interference from uric acid and acetaminophen. The lactate sensors were adequately stable for at least 48 days of storage at 25 °C. These results indicated that mPES can be replaced with 1-methoxy-5-methyl phenazinium methyl sulfate (mPMS), which we previously reported as the best mediator for AvLOx-based lactate sensors. Furthermore, this study revealed that mPES can be used as an effective electron mediator for the enzyme sensors employing representative flavin oxidoreductases, GDH-based glucose sensors, and FPOx-based hemoglobin A1c (HbA1c) sensors.

G-quadruplex-forming GGA repeat region functions as a negative regulator of the Ccnb1ip1 enhancer.

An enhancer located upstream of the transcriptional start site of Ccnb1ip1 containing two GGA-rich regions and a 14-GGA repeat (GGA)14 region has been previously identified. Three copies of four GGA repeats in the c-myb promoter that form a tetrad:heptad:heptad:tetrad (T:H:H:T) dimerized G-quadruplex (G4) structure reportedly functions as both a transcriptional repressor and activator. Here, the secondary structures of the two GGA-rich and (GGA)14 regions were analyzed using circular dichroism spectral analysis, which indicated that the two GGA-rich DNAs formed parallel-type G4 structures, whereas (GGA)14 DNA formed the T:H:H:T dimerized G4 structure. Reporter assays demonstrated that individual regions did not show enhancer activity; however, the deletion of the (GGA)14 region resulted in 1.5-fold higher enhancer activity than that of the whole enhancer. These results indicate that the (GGA)14 region that forms the T:H:H:T dimerized G4 structure functions as a negative regulator of the Ccnb1ip1 enhancer.