Tuning CpG motif position in nanostructured DNA for efficient immune stimulation.

It was previously demonstrated that polypod-like nanostructured DNA (polypodna) comprising three or more oligodeoxynucleotides (ODNs) were useful for the delivery of ODNs containing cytosine-phosphate-guanine (CpG) motifs, or CpG ODNs, to immune cells. Although the immunostimulatory activity of single-stranded CpG ODNs is highly dependent on CpG motif sequence and position, little is known about how the position of the motif affects the immunostimulatory activity of CpG motif-containing nanostructured DNAs. In the present study, four series of polypodna were designed, each comprising a CpG ODN with one potent CpG motif at varying positions and 2-5 CpG-free ODNs, and investigated their immunostimulatory activity using Toll-like receptor-9 (TLR9)-positive murine macrophage-like RAW264.7 cells. Polypodnas with the CpG motif in the 5'-overhang induced more tumor necrosis factor-α release than those with the motif in the double-stranded region, even though their cellular uptake were similar. Importantly, the rank order of the immunostimulatory activity of single-stranded CpG ODNs changed after their incorporation into polypodna. These results indicate that the CpG ODN sequence as well as the motif location in nanostructured DNAs should be considered for designing the CpG motif-containing nanostructured DNAs for immune stimulation.

Exploring the formation mechanism of ferulic acid/hydroxypropyl-ß-cyclodextrin inclusion complex: spectral analyses and computer simulation.

BACKGROUND: The biological functions of ferulic acid (FA) have garnered significant interest but its limited solubility and stability have led to low bioavailability. Hydroxypropyl-ß-cyclodextrin (HP-ß-CD), with its distinctive hollow structure, offers the potential for encapsulating hydrophobic molecules. The formation of an inclusion complex between FA and HP-ß-CD may therefore be a viable approach to address the inherent limitations of FA. To investigate the underlying mechanism of the FA/HP-ß-CD inclusion complex formation, a combination of spectral analyses and computer simulation was employed. RESULTS: The disappearance of the characteristic peaks of FA in Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) confirmed the formation of an inclusion complex between FA and HP-ß-CD. Thermogravimetry-derivative thermogravimetry (TG-DTG) studies demonstrated that the thermal stability of FA was enhanced due to the encapsulation of FA within HP-ß-CD. Molecular dynamics simulation also provided evidence that FA successfully penetrated the HP-ß-CD cavity, primarily driven by van der Waals interactions. The formation of the complex resulted in more compact HP-ß-CD structures. The bioavailability of FA was also strengthened through the formation of inclusion complexes with HP-ß-CD. CONCLUSIONS: The findings of this study have contributed to a deeper understanding of the interactions between FA and HP-ß-CD, potentially advancing a delivery system for FA and enhancing the bioavailability of insoluble active components. © 2024 Society of Chemical Industry.

Geographical Origin Differentiation of Rice by LC-MS-Based Non-Targeted Metabolomics.

Many factors, such as soil, climate, and water source in the planting area, can affect rice taste and quality. Adulterated rice is common in the market, which seriously damages the production and sales of high-quality rice. Traceability analysis of rice has become one of the important research fields of food safety management. In this study, LC-MS-based non-targeted metabolomics technology was used to trace four rice samples from Heilongjiang and Jiangsu Provinces, namely, Daohuaxiang (DH), Huaidao No. 5 (HD), Songjing (SJ), and Changlixiang (CL). Results showed that the discrimination accuracy of the partial least squares discriminant analysis (PLS-DA) model was as high as 100% with satisfactory prediction ability. A total of 328 differential metabolites were screened, indicating significant differences in rice metabolites from different origins. Pathway enrichment analysis was carried out on the four rice samples based on the KEGG database to determine the three metabolic pathways with the highest enrichment degree. The main biochemical metabolic pathways and signal transduction pathways involved in differential metabolites in rice were obtained. This study provides theoretical support for the geographical origins of rice and elucidates the change mechanism of rice metabolic pathways, which can shed light on improving rice quality control.

Lilium pumilum stress-responsive NAC transcription factor LpNAC17 enhances salt stress tolerance in tobacco.

Lilium pumilum is a perennial herb with ornamental edible and medicinal value. It is an excellent wild germplasm resource with wide distribution and strong resistance. The NAC family of transcription factors is unique to higher plants. The NAC family plays a regulatory role in plant growth and development and participates in plant responses to biotic and abiotic stresses. The LpNAC17 gene of L. pumilum was cloned and transformed into tobacco to investigate the response of transgenic tobacco to salt stress. The results showed that the net photosynthetic rate and contents of chlorophyll in LpNAC17 over-expressed tobacco were higher than those in the control plants, while the stomatal conductance, transpiration rate and intercellular CO2 concentration were lower than those in the controls. The activity of superoxide dismutase, peroxidase, catalase, and the content of proline in LpNAC17 over-expressed tobacco were higher than those in the controls, while the content of malondialdehyde, superoxide anion, and hydrogen peroxide were lower than that in the control. Nitro-blue tetrazolium staining and 3,3'-diaminobenzidine tissue localization showed that the contents of O 2 - and H2O2 in transgenic tobacco was lower than in the controls. The expression levels of NtSOD, NtPOD, NtCAT, NtHAK1, NtPMA4, and NtSOS1 in the transgenic tobacco were higher than those in the controls. Therefore, this study provides a gene source for molecular breeding of salt-tolerant plants through genetic engineering, and lays a foundation for further research on salt-tolerant Lily.

LpNAC6 reversely regulates the alkali tolerance and drought tolerance of Lilium pumilum.

NAC transcription factors have multiple biological functions in plants. In this study, a new NAC transcription factor, LpNAC6, was cloned from Lilium pumilum, and its salt and drought resistance functions were identified. We treated LpNAC6 transgenic tobacco plants with different intensities of alkali and drought stress. Results showed that LpNAC6 transgenic tobacco had enhanced alkali tolerance, but decreased drought tolerance. Antioxidant enzyme (SOD, POD, CAT) activity, chlorophyll content, proline content, and photosynthetic capacity of transgenic tobacco were significantly higher than those of wild-type tobacco, while the contents of MDA, H2O2, and O2- were significantly lower than those of wild-type tobacco. The expression level of stress-related genes in transgenic tobacco increased significantly, and the alkali tolerance was enhanced, but the opposite was true under drought stress. Our findings suggest that LpNAC6 has a reverse regulatory effect on alkaline and drought tolerance in plants, which is of great significance for plant screening and stress tolerance regulation of transgenic plants in arid saline-alkali land.

Construction of Monomeric and Dimeric G-Quadruplex-Structured CpG Oligodeoxynucleotides for Enhanced Uptake and Activation in TLR9-Positive Macrophages.

The G-quadruplex (GQ) structure has potential applications in nucleic acid drug delivery because of its superior stability. In this study, we added one G-tract (five guanines) to an unmethylated phosphodiester-linked cytosine-phosphate-guanine oligodeoxynucleotide (CpG ODN), a potential immune adjuvant, to construct a GQ-structured CpG ODN with precise structural properties, increased biological stability, and efficient delivery to Toll-like receptor 9 (TLR9)-positive immune cells. A G-tract was added to phosphodiester-backboned CpG1668 at the 5'-end [1668(5'-G5)], 3'-end [1668(3'-G5)], or within the sequence [1668(mid-G5)]. Circular dichroism analysis showed that all CpG ODNs with a G-tract formed parallel GQ structures, irrespective of its position. Electrophoresis showed that 1668(5'-G5) formed a GQ dimer, whereas others remained GQ monomers. GQ-structured CpG ODNs induced greater tumor necrosis factor-α and interleukin-6 secretion from TLR9-positive mouse macrophage-like RAW264.7 cells than single-stranded CpG ODNs, with the highest for 1668(3'-G5). GQ structuration increased CpG ODN uptake by RAW264.7 cells, and 1668(3'-G5) decomposed more slowly in serum than 1668(5'-G5). Thus, GQ formation with one G-tract is a simple and efficient strategy for CpG ODN delivery to TLR9-positive cells, and addition of a G-tract to the 3'-end is effective in obtaining monomeric GQ-structured CpG ODN with high biological stability and immunostimulatory activity.

Analysis of Tertiary Structural Features of Branched DNA Nanostructures with Partially Common Sequences Using Small-Angle X-ray Scattering.

We demonstrated that polypod-like structured DNA, composed of 3-8 kinds of oligodeoxynucleotides (ODNs), exhibit accelerated cellular uptake depending on their structural properties. The biological activities, including immunostimulation and unwanted adverse effects of ODNs depend on their sequences. Therefore, as the number of different types of ODNs within a polypod-like structured DNA increases, the possible risks and concerns regarding its future clinical applications also increase. To minimize this risk and to explore the relationship between structural properties and cellular uptake, we designed Tet(id12), a tetrapod-like structured DNA with 12 identical palindrome sequences in the center of ODNs, and compared it with other tetrapod-like structures without palindrome sequences, symmetric Tet(sym) and asymmetric Tet(asym12). The thermal stability analysis of Tet(id12) revealed a two-step dissociation process. Polyacrylamide gel electrophoresis, small-angle X-ray scattering profile, and the Guinier plot also revealed that the conformation of Tet(id12) was close to that of Tet(sym). No significant differences were observed between Tet(id12) and others in terms of cellular uptake. These results indicate that ODNs with palindrome sequences can be used to design polypod-like structured DNAs without significant changes to their physicochemical and biological properties, although their thermal stability is somewhat different from the ones without palindrome sequences.

Nanostructured DNA for the delivery of therapeutic agents.

DNA and RNA, the nucleic acids found in every living organism, are quite crucial, because not only do they store the genetic information, but also they are used as signals through interaction with various molecules within the body. The nature of nucleic acids, especially DNA, to form double-helix makes it possible to design nucleic acid-based nanostructures with various shapes. Because the shapes as well as the physicochemical properties determine their interaction with proteins or cells, nanostructured DNAs will have different features in the interaction compared with single- or double-stranded DNA. Some of these unique features of nanostructured DNA make ways for efficient delivery of therapeutic agents to specific targets. In this review, we begin with the factors affecting the properties of nanostructured DNA, followed by summarizing the methods for the development of nanostructured DNA. Further, we discuss the characteristics of nanostructured DNA and their applications for the delivery of bioactive compounds.

DNA nanotechnology-based composite-type gold nanoparticle-immunostimulatory DNA hydrogel for tumor photothermal immunotherapy.

Success of tumor photothermal immunotherapy requires a system that induces heat stress in cancer cells and enhances strong anti-tumor immune responses. Here, we designed a composite-type immunostimulatory DNA hydrogel consisting of a hexapod-like structured DNA (hexapodna) with CpG sequences and gold nanoparticles. Mixing of the properly designed hexapodna and oligodeoxynucleotide-modified gold nanoparticles resulted in the formation of composite-type gold nanoparticle-DNA hydrogels. Laser irradiation of the hydrogel resulted in the release of hexapodna, which efficiently stimulated immune cells to release proinflammatory cytokines. Then, EG7-OVA tumor-bearing mice received an intratumoral injection of a gold nanoparticle-DNA hydrogel, followed by laser irradiation at 780 nm. This treatment increased the local temperature and the mRNA expression of heat shock protein 70 in the tumor tissue, increased tumor-associated antigen-specific IgG levels in the serum, and induced tumor-associated antigen-specific interferon-γ production from splenocytes. Moreover, the treatment significantly retarded the tumor growth and extended the survival of the tumor-bearing mice.

Polypod-Shaped DNAs: Small-Angle X-ray Scattering and Immunostimulatory Activity.

We explored in detail the relationship between the structure in aqueous solution and immunostimulatory activity of polypod-shaped DNAs, called polypodnas. The polypodnas were constructed using 3-6 oligodeoxynucleotides (ODNs) to obtain tri-, tetra-, penta-, and hexapodna, each of which had 3, 4, 5, and 6 arms made of double-stranded DNA, respectively. A highly potent immunostimulatory CpG sequence was included into each of the polypodnas. Synchrotron X-ray scattering analysis showed that the double-stranded DNA arms of all of the polypodnas adopted a B-form DNA conformation. The analysis also suggested that some nucleotides in the central parts of pentapodna and hexapodna did not form base pairs, whereas those of tripodna and tetrapodna all formed base pairs. This difference would occur because of an increase in steric hindrance and electrical repulsion with increasing number of arms. The pentapodna and hexapodna induced a large amount of tumor necrosis factor α-release from macrophage-like cells compared with the tripodna and tetrapodna, suggesting that the partly loosened DNA in polypodna with many arms is advantageous for exposing the immunostimulatory sequences of the polypodna.

Efficient amplification of self-gelling polypod-like structured DNA by rolling circle amplification and enzymatic digestion.

The application of DNA as a functional material such as DNA hydrogel has attracted much attention. Despite an increasing interest, the high cost of DNA synthesis is a limiting factor for its utilization. To reduce the cost, we report here a highly efficient amplification technique for polypod-like structured DNA (polypodna) with adhesive ends that spontaneously forms DNA hydrogel. Two types of polypodna with three (tripodna) and four (tetrapodna) pods were selected, and a template oligodeoxynucleotide, containing a tandem sequence of a looped tripodna or tetrapodna, respectively, along with restriction enzyme (TspRI) sites, was designed. The template was circularized using T4 DNA ligase, and amplified by rolling circle amplification (RCA). The RCA product was highly viscous and resistant to restriction digestion. Observation under an electron microscope revealed microflower-like structures. These structures were composed of long DNA and magnesium pyrophosphate, and their treatment with EDTA followed by restriction digestion with TspRI resulted in numerous copies of polypodna with adhesive ends, which formed a DNA hydrogel. Thus, we believe this technique provides a new approach to produce DNA nanostructures, and helps in expanding their practical applications.

Risk factors of recurrent aphthous ulceration among university students.

Recurrent aphthous ulceration (RAU) is a common oral mucosal disease. The etiological involves in genetics, vitamin deficiencies, trauma, immune dysfunction and stress. This study was to explore the related risk factors of recurrent aphthous ulceration (RAU) among college students, and provide basis for further research. We conducted a questionnaire survey among students from three colleges in Wuhu by stratified cluster sampling. The information collected includes general demographic characteristics, dietary habits and so on. The overall prevalence of RAU is 23.30% among college students (23.23% in male and 23.39% in female). There are statistical significance in prevalence of RAU between subjects with RAU and without RAU (P<0.05) the prevalence of RAU in different grade, age, adequate brushing time, good brushing habits, wear dentures or braces, other oral disease, eat barbecue, adequate exercise time is statistic difference. According to the result of multinomial logistic regression analysis, the risk of recurrent aphthous ulceration factors including grade, inadequate brushing time. Tempering was a protective factor of RAU. Some measure should be taken to control dental ulcer, which consist of promoting a correct way of living habits, paying attention to the health conscious diet, strengthen physical exercise, self-decompression and keeping good mentality.