Coarse-grained model simulation-guided localized DNA signal amplification probe for miRNA detection.

DNA-based enzyme-free signal amplification strategies are widely employed to detect biomarkers in low abundance. To enhance signal amplification, localized DNA reaction units which increases molecular collision probability is commonly utilized. However, the current understanding of the structure-function relationships in localized DNA signal amplification probes is limited, leading to unsatisfied performance. In this study, we introduced a coarse-grained molecular model to simulate the dynamic behavior of two DNA reaction units within a DNA enzyme-free signal amplification circuit called Localized Catalytic Hairpin Assembly (LCHA). We investigated the impact of localized distance and flexibility on reaction performance. The most efficient LCHA probe guided by simulation exhibits sensitivity 28 times greater that of free CHA, with a detection limit of miR-21 reaching 16 pM, while the least effective LCHA probe demonstrated a modest improvement of only 7 times. We successfully employed the optimized probe to differentiate cancer cells from normal cells based on their miR-21 expression levels, showcasing its quantification ability. By elucidating the mechanistic insights and structure-function relationship in our work, we aim to contribute valuable information that can save users' time and reduce costs when designing localized DNA probes. With a comprehensive understanding of how the localization affects probe performance, researchers can now make more informed and efficient decisions during the design process. This work would find broad applications of DNA nanotechnology in biosensing, biocomputing, and bionic robots.

Comparative Transcriptome Analysis of High- and Low-Growth Genotypes of Eucalyptus urophylla in Response to Long-Term Nitrogen Deficiency.

Nutrients play important roles in the growth and development of most plant species. However, in perennial trees, the function of nutrients in different genotypes is poorly understood. Three different nutrient levels (low, sufficient, and high nutrient levels) were applied to two contrasting Eucalyptus urophylla cultivars (a high-growth cultivar ZQUA44 and a low-growth cultivar ZQUB15), and growth and expression levels were analyzed. Although the growth traits of both genotypes under nutrient starvation treatment were much lower than under abundant nutrients, tree height, crown width, and biomass of different ZQUA44 tissues were much higher than those of ZQUB15 at all three nutrient levels. Differentially expressed genes (DEGs) clustered into six subclusters based on their expression patterns, and functional annotation showed that the DEGs involved in glutathione metabolism and flavonoid biosynthesis may be responsible for nutrient starvation across different genotypes, while the DEGs involved in carotenoid biosynthesis and starch and sucrose metabolism may have a range of functions in different genotypes. The DEGs encoding the MYB-related family may be responsible for nutrient deficiency in all genotypes, while B3 may have different functions in different genotypes. Our results demonstrate that different genotypes may form different pathways to coordinate plant survival when they face abiotic stresses.

Integrated transcriptomic and gibberellin analyses reveal genes related to branch development in Eucalyptus urophylla.

Tree branches affect the planting density and basal scab, which act as important attributes in the yield and quality of trees. Eucalyptus urophylla is an important pioneer tree with characteristics of strong adaptability, fast growth, short rotation period, and low disease and pest pressures. In this study, we collected ZQUC14 and LDUD26 clones and compared their transcriptomes and metabolomes from mature xylem, phloem, and developing tissues to identify factors that may influence branch development. In total, 32,809 differentially expressed genes (DEGs) and 18 gibberellin (GA) hormones were detected in the five sampled tissues. Searches of the kyoto Encyclopedia of Genes and Genomes pathways identified mainly genes related to diterpenoid biosynthesis, plant MAPK signaling pathways, plant hormone signal transduction, glycerolipid metabolism, peroxisome, phenylpropanoid biosynthesis, ABC transporters, and brassinosteroid biosynthesis. Furthermore, gene expression trend analysis and weighted gene co-expression network analysis revealed 13 genes likely involved in diterpenoid biosynthesis, including five members of the 2OG-Fe(II) oxygenase superfamily, four cytochrome P450 genes, and four novel genes. In GA signal transduction pathways, 24 DEGs were found to positively regulate branch formation. These results provide a comprehensive analysis of branch development based on the transcriptome and metabolome, and help clarify the molecular mechanisms of E. urophylla.

A new self-attenuated therapeutic influenza vaccine that uses host cell-restricted attenuation by artificial microRNAs.

New strategies are urgently needed for developing vaccines and/or anti-viral drugs against influenza viruses, because antigenic shift and drift inevitably occurs in circulating strains each year, and new strains resistant to anti-viral drugs have recently emerged. In our study, we designed and incorporated artificial microRNAs (amiRNAs) into the NA segment of rescued influenza viruses to separately target two host genes, Cdc2-like kinase 1 (CLK1) and SON DNA binding protein (SON), which were found to play an essential role in virus replication. Mouse epithelial fibroblast (MEF) or human lung carcinoma A549 cells infected with engineered influenza PR8 viruses expressing amiR-30CLK1 (PR8-amiR-30CLK1) or amiR-93SON (PR8-amiR-93SON) had reduced expression of host proteins CLK1 and SON, respectively. All engineered influenza viruses functioned as attenuated vaccines, induced significantly higher antibody responses, and provided greater protective efficacy. In addition, they were found to be safe, based on the mouse weight changes and clinical signs observed. In contrast to the engineered viruses targeting SON, mice treated with engineered viruses targeting CLK1 recovered from weight loss and survived lethal infection by 6 h after lethal-dose PR8 infection, suggesting that our PR8-amiR-30CLK1 self-attenuated influenza virus (SAIV) could be used as a new therapeutic influenza vaccine.

CGFe and TGF-ß1 enhance viability and osteogenic differentiation of human dental pulp stem cells through the MAPK pathway.

The present study aimed to evaluate the effects of concentrated growth factor exudate (CGFe) and TGF-ß1 on the viability and osteogenic differentiation of human dental pulp stem cells (hDPSCs). CGFe was prepared from the peripheral blood of healthy donors (obtained with informed consent). STRO-1+ hDPSCs were isolated from dental pulp tissues and treated in four groups: i) Control; ii) TGF-ß1 (1 ng/ml); iii) 100% CGFe; and iv) TGF-ß1 (1 ng/ml) + 100% CGFe group. hDPSC viability was measured via MTT assay. The osteogenic differentiation of hDPSCs was quantified via alkaline phosphatase (ALP) activity, western blotting and reverse transcription-quantitative PCR assays. CGFe and TGF-ß1 enhanced hDPSC viability, upregulated ALP activity, upregulated the expression of phosphorylated (p)-ERK1/2, p-JNK and p-p38 in hDPSCs, and promoted transcription and protein expression of osteogenic-related genes (bone sialoprotein, Runt-related transcription factor 2 and osteocalcin) in hDPSCs. The present study demonstrated that CGFe and TGF-ß1 facilitated the viability and osteogenic differentiation of hDPSCs potentially through activation of the MAPK signaling pathway.

Transcriptome and metabolite analysis related to branch development in two genotypes of Eucalyptus urophylla.

Branching in long-lived plants can cause scarring at the base and affect wood density, which greatly inhibits wood yield and quality. Eucalyptus urophylla is one of the most important commercial forest tree species in South China, with diverse branch number and branch angles under different genetic backgrounds. However, the main elements and regulatory mechanisms associated with different branching traits in E. urophylla remain unclear. To identify the factors that may influence branching, the transcriptome and metabolome were performed on the shoot apex (SA), lateral shoot apex (LSA), and stem segment at the 5th axillary bud from the shoot apex (S1) in lines ZQUC14 (A) and LDUD26 (B), with A exhibiting a smaller Ba than B. A total of 307.3 million high-quality clean reads and nine hormones were identified from six libraries. Several differentially expressed regulatory factors were identified between the two genotypes of E. urophylla. The Kyoto Encyclopedia of Genes and Genomes pathways were enriched in plant hormone signal transduction, plant hormone biosynthesis and their transport pathways. Furthermore, gene expression pattern analysis identified genes that were significantly downregulated or upregulated in S1 relative to the SA and LSA segments, and the plant hormone signal transduction pathway was constructed to explain branching development. This study clarified the main plant hormones and genes underlying branch numbers and angles of E. urophylla, confirmed that ABA and SA could promote a larger branch angle and smaller branch number, while IAA has an opposite function. Numbers of key candidate genes involved in plant hormone signal transduction were found in the positive regulation of branch formation. These novel findings should aid molecular breeding of branching in Eucalyptus.

Distribution of ammonia-oxidizing archaea and bacteria along an engineered coastal ecosystem in subtropical China.

Ammonia-oxidizing archaea (AOA) and bacteria (AOB) are the crucial players in nitrogen cycle. Both AOA and AOB were examined along a gradient of human activity in a coastal ecosystem from intertidal zone, grassland, and Casuarina equisetifolia forest to farmland. Results showed that the farmland soils had noticeably higher nitrate-N, available P than soils in the other three sites. Generally, AOA and AOB community structures varied across sites. The farmland mainly had Nitrosotalea-like AOA, intertidal zone was dominated by Nitrosopumilus AOA, while grassland and C. equisetifolia forest primarily harbored Nitrososphaera-like AOA. The farmland and C. equisetifolia forest owned Nitrosospira-like AOB, intertidal zone possessed Nitrosomonas-like AOB, and no AOB was detected in the grassland. AOA abundance was significantly greater than AOB in this coastal ecosystem (p < 0.05, n = 8). AOB diversity and abundance in the farmland were significantly higher than those in the other three sites (p < 0.05, n = 2). The biodiversity and abundance of AOA were not significantly correlated with any soil property (p < 0.05, n = 8). However, the diversity of AOB was significantly correlated with pH, available P and total P (p < 0.05, n = 6). The abundance of AOB was significantly correlated with pH, nitrite, available N, available P and total P (p < 0.05, n = 6). This study suggested that the community structures of AOA and AOB vary in the different parts in the bio-engineered coastal ecosystem and agricultural activity appears to influence these nitrifiers.

Genome-Wide Analysis of Basic Helix-Loop-Helix Transcription Factors to Elucidate Candidate Genes Related to Fruit Ripening and Stress in Banana (Musa acuminata L. AAA Group, cv. Cavendish).

The basic helix-loop-helix (bHLH) proteins are a superfamily of transcription factors (TFs) that can bind to specific DNA target sites, playing a central role in a wide range of metabolic, physiological, and developmental processes in higher organisms. However, no systemic analysis of bHLH TFs has been reported in banana, a typical climacteric fruit in tropical and subtropical regions. In our study, 259 MabHLH TF genes were identified in the genome of Musa acuminata (A genome), and phylogenetic analysis indicated that these MabHLHs could be classified into 23 subfamilies with the bHLHs from rice and Arabidopsis. The amino acid sequences of the bHLH domain in all MabHLH protein sequences were quite conserved, especially Arg-12, Arg-13, Leu-23, and Leu-79. Distribution mapping results showed that 258 MabHLHs were localized on the 11 chromosomes in the M. acuminata genome. The results indicated that 40.7% of gene duplication events were located in collinear fragments, and segmental duplications might have played a key role in the expansion of MabHLHs. Moreover, the expression profiles of MabHLHs in different fruit development and ripening stages and under various abiotic and biotic stresses were investigated using available RNA-sequencing data to obtain fruit development, ripening-specific, and stress-responsive candidate genes. Finally, a co-expression network of MabHLHs was constructed by weighted gene co-expression network analysis to elucidate the MabHLHs that might participate in important metabolic biosynthesis pathways in banana during development and the response to stress. A total of 259 MabHLHs were identified, and their sequence features, conserved domains, phylogenetic relationships, chromosomal distributions, gene duplications, expression profiles, and co-expression networks were investigated. This study systematically identified the MabHLHs in the M. acuminata genome at the genome-wide level, providing important candidate genes for further functional analysis. These findings improve our understanding of the molecular basis of developmental and stress tolerance in an important banana cultivar.

Anti-caries vaccine based on clinical cold-adapted influenza vaccine: A promising alternative for scientific and public-health protection against dental caries.

Dental caries remains one of the most pervasive infectious disease around the world. Protection against dental caries can be achieved experimentally by eliciting salivary IgA targeting surficial antigens of S. mutans, however, no such a vaccine has been launched for human use yet. Live vectored vaccines hold the greatest feasibility to induce potent and long-lasting immunity in the host. The FDA approved intranasal cold-adapted influenza vaccine has been used in clinical settings for many years. The vaccine can not only induce broad adaptive immune responses especially mucosal immunity, but the member strains can also circumvent existing immunity, presenting promising candidates for live vectored anti-caries vaccine. Moreover, the genetic techniques for modification of cold-adapted influenza viruses are well developed and widely applicable. Thus, we hypothesize that effective anti-caries vaccine can be developed with the backbone of cold-adapted influenza viruses by inserting specific antigenic identifier sequences of S. mutans into the viral genome, which is anticipated to protect against dental caries in humans with easy inoculation. The immune efficacies of recombinant cold-adapted influenza viruses expressing exogenous antigens have been verified by in vivo experiments for multiple infectious diseases, giving us great confidence to validate the safety properties and protection effect with this chimeric vaccine in animals or even humans. Existing data suggests that the live anti-caries vaccine may help improve public oral health by controlling the caries disease itself.

Concentrated growth factor exudate enhances the proliferation of human periodontal ligament cells in the presence of TNF­α.

The purpose of this study was to evaluate the effects of concentrated growth factor exudate (CGFe) on human periodontal ligament cells (hPDLCs) stimulated by tumor necrosis factor (TNF)­α. From the peripheral blood of healthy donors, CGFe was prepared according to the Sacco protocol after 7 days of incubation. The hPDLCs were cultured by a tissue explant method and identified with anti­vimentin and anti­cytokeratin antibodies. Cells were subjected to four different treatments: i) Control; ii) TNF­α (10 ng/ml); iii) CGFe (concentration 50%); and iv) CGFe+TNF­α. The proliferation of hPDLCs was measured with Cell Counting Kit­8 assays. Osteogenic differentiation and mineralization were determined by Alizarin Red S staining, alkaline phosphatase activity, western blotting and reverse transcription­quantitative polymerase chain reaction. CGFe enhanced cell proliferation and upregulated ALP activity, the mineralization level, and osteogenic­associated osteocalcin, runt­related transcription factor 2 and Osterix gene expression in hPDLCs under inflammatory conditions induced by TNF­α. The present study demonstrated that CGFe enhanced hPDLC proliferation and osteogenic differentiation in the presence of TNF­α­induced inflammation. As CGFe can be obtained from the venous blood of patients, it generates no immune reaction. Thus, it is more economical and beneficial to use CGFe in clinical periodontal regeneration practice than synthetic growth factors.

Platelet­rich fibrin exudate promotes the proliferation and osteogenic differentiation of human periodontal ligament cells in vitro.

The purpose of the present study was to evaluate the effects of platelet­rich fibrin (PRF) exudate on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells (hPDLCs) in vitro. In the present study PRF was obtained with permission, from the peripheral blood of healthy donors and PRF exudates were collected on the 7th day of incubation. hPDLCs were obtained from healthy premolars, cultured by a tissue explant method and identified with anti­vimentin and anti­cytokeratin antibodies. PRF exudates were added to hPDLCs in different concentrations to evaluate cell proliferation and osteogenic differentiation. The proliferation of hPDLCs was measured using a colorimetric assay. Osteogenic differentiation and mineralization were determined by Alizarin red staining, alkaline phosphatase activity (ALP), western blotting and reverse transcription­quantitative polymerase chain reaction. Cell proliferation was enhanced by addition of the PRF exudate, which also promoted the formation of mineralized matrix nodules and upregulated ALP activity and osteoblast­associated levels of osteocalcin, runt­related transcription factor and osterix gene expression. As these stimulatory effects occurred in a dose­dependent manner, it was concluded that high concentrations of the PRF exudate served an essential role in the proliferation, osteogenic differentiation and mineralization of hPDLCs in vitro. The present study demonstrated that PRF exudate enhanced hPDLC proliferation, induced the osteoblastic differentiation of hPDLCs into mineralized tissue­formation cells in vitro, and may therefore provide potential benefits for periodontal tissue engineering; contributing to the primary processes of periodontal tissue regeneration. From the perspective of both economics and biology, PRF has greater clinical benefits than analogous growth factors.

Genetic diversity and phylogenetic relationships of seven Amorphophallus species in southwestern China revealed by chloroplast DNA sequences.

Plants species in the genus Amorphophallus are of great economic importance, as they are the only plants known to produce glucomannan. Although southwestern China has been recognized as one of the origin centres of Amorphophallus, only a few studies assessing its genetic diversity have been reported. To aid in the utilization and conservation of Amorphophallus species, we evaluated the genetic diversity and phylogenetic relationships among seven edible Amorphophallus species using three chloroplast DNA regions (rbcL, trnL and trnK-matK). The results showed that the genetic diversity at the population level was relatively low, with over half of the populations harbouring only one haplotype. The widely scattered species, A. konjac, had the largest genetic diversity, while the narrow endemic species, A. yuloensis, possessed only one haplotype. Phylogeny analysis identified three well-supported major lineages. Our study suggested that habitat fragmentation might be a driver of the genetic variation patterns within and between populations of Amorphophallus. A conservation strategy consisting of in situ conservation and germplasm collection is recommended.

An overwhelmingly selective colorimetric sensor for Ag(+) using a simple modified polyacrylonitrile fiber.

A carboxymethyl-dithiocarbamate immobilized polyacrylonitrile fiber colorimetric sensor has been synthesized. This fiber sensor exhibits excellent selectivity and sensitivity for Ag(+) in aqueous solution with a remarkable color change from light pink to red-brown over a wide pH range of 2-12. The sensor responds selectively to Ag(+) in the presence of other ions, including Mg(2+), Al(3+), Ca(2+), Cr(3+), Mn(2+), Co(2+), Ni(2+), Cu(2+), Zn(2+), Cd(2+), Hg(2+) and Pb(2+). The colorimetric sensor has an extremely fast response time (10s) and a low visual limit of detection (5.53×10(-12) mol/L). The fiber sensor also undergoes an obvious color change in the presence of Ag(+) solutions containing EDTA, NaCl or NaBr. Density functional theory optimization reveals that the sensor and Ag(+) interact via a seven-membered ring complexation mechanism.