Accurate identification of single nucleotide variants (SNVs) in key driver genes holds a significant value for disease diagnosis and treatment. Fluorescent probes exhibit tremendous potential in specific, high-resolution, and rapid detection of SNVs. However, additional steps are required in most post-PCR assays to convert double-stranded DNA (dsDNA) products into single-stranded DNA (ssDNA), enabling them to possess hybridization activity to trigger subsequent reactions. This process not only prolongs the complexity of the experiment but also introduces the risk of losing target information. In this study, we proposed two strategies for enriching active double-stranded DNA, involving PCR based on obstructive groups and cleavable units. Building upon this, we explored the impact of modified units on the strand displacement reaction (SDR) and assessed their discriminatory efficacy for mutations. The results showed that detection of low variant allele frequencies (VAF) as low as 0.1% can be achieved. The proposed strategy allowed orthogonal identification of 45 clinical colorectal cancer tissue samples with 100% specificity, and the results were generally consistent with sequencing results. Compared to existing methods for enriching active targets, our approach offers a more diverse set of enrichment strategies, characterized by the advantage of being simple and fast and preserving original information to the maximum extent. The objective of this study is to offer an effective solution for the swift and facile acquisition of active double-stranded DNA. We anticipate that our work will facilitate the practical applications of SDR based on dsDNA.
DNA , Polymorphism, Single Nucleotide , Polymorphism, Single Nucleotide/genetics , Humans , DNA/genetics , DNA/chemistry , Colorectal Neoplasms/genetics , Polymerase Chain Reaction , Fluorescent Dyes/chemistry , DNA, Single-Stranded/genetics , DNA, Single-Stranded/chemistry
Accurate microRNA (miRNA) detection is pivotal in the diagnosis and monitoring of cancer. Entropy-driven catalysis (EDC) has attracted widespread attention as an enzyme-free, isothermal technique for miRNA detection owing to its inherent simplicity and reliability. However, conventional EDC is a single-output mode, limiting the efficiency of signal amplification. In this study, a novel EDC dual-output mode was employed in conjunction with DNAzyme, resulting in the development of an EDC dual-end DNAzyme (EDC-DED) approach for highly sensitive miRNA detection. In this system, miRNA-21 initiated the EDC reaction, producing a large amount of catalytically active dual-end Mg2+-dependent DNAzyme. The DNAzyme further cleaved the reporter cyclically, generating a notably amplified fluorescence signal. The proposed method achieved a low detection limit of 2 pM. Compared with the traditional EDC single-end DNAzyme (EDC-SED) strategy, the present method exhibited superior amplification efficiency, enhancing detection sensitivity by approximately 46.5-fold. Furthermore, this platform demonstrated ideal specificity, satisfactory reproducibility and acceptable detection capabilities in clinical serum samples. Therefore, the straightforward and convenient strategy is a potential tool for miRNA analysis, which may provide a new perspective for biological analysis and clinical application.
Understanding the molecular mechanisms that regulate flower growth, development, and opening is of paramount importance, yet these processes remain less explored at the genetic level. Flower development in Hydrangea paniculata 'Vanilla Strawberry' is finely tuned through hormonal signals, yet the genetic underpinnings are not well defined. This study addresses the gap by examining the influence of gibberellic acid (GA3), salicylic acid (SA), and ethylene (ETH) on the flowering traits and underlying molecular responses. Treatment with 100 mg/L SA significantly improved chlorophyll content and bolstered the accumulation of soluble sugars and proteins, advancing the flowering onset by 6 days and lengthening the flowering period by 11 days. Concurrently, this treatment enhanced inflorescence dimensions, increasing length, width, and petal area by 22.76%, 26.74%, and 27.45%, respectively. Contrastingly, 100 mg/L GA3 expanded inflorescence size but postponed flowering initiation and decreased inflorescence count. Higher concentrations of SA and GA3, as well as any concentration of ETH, resulted in delayed flowering and inferior inflorescence attributes. A physiological analysis over 50 days revealed that these regulators variably affected sugar and protein levels and modified antioxidant enzyme activities. An RNA-seq analysis during floral development highlighted significant transcriptomic reprogramming, with SA treatment downregulating Myb transcription factors, implicating them in the modulation of flowering timing and stress adaptation. These findings illuminate the complex interplay between hormonal treatments, gene expression, and flowering phenotypes in Hydrangea paniculata, offering valuable perspectives for ornamental horticulture optimization.
DNA strand displacement reactions are vital for constructing intricate nucleic acid circuits, owing to their programmability and predictability. However, the scarcity of effective methods for eliminating circuit leakages has hampered the construction of circuits with increased complexity. Herein, a versatile strategy is developed that relies on a spatially controlled proximity split tweezer (PST) switch to transduce the biomolecular signals into the independent oligonucleotides. Leveraging the double-stranded rigidity of the tweezer works synergistically with the hindering effect of the hairpin lock, effectively minimizing circuit leakage compared with sequence-level methods. In addition, the freely designed output strand is independent of the target binding sequence, allowing the PST switch conformation to be modulated by nucleic acids, small molecules, and proteins, exhibiting remarkable adaptability to a wide range of targets. Using this platform, established logical operations between different types of targets for multifunctional transduction are successfully established. Most importantly, the platform can be directly coupled with DNA catalytic circuits to further enhance transduction performance. The uniqueness of this platform lies in its design straightforwardness, flexibility, scalable intricacy, and system compatibility. These attributes pave a broad path toward nucleic acid-based development of sophisticated transduction networks, making them widely applied in basic science research and biomedical applications.
Coronary heart disease (CHD) is one of the most serious public health problems. However, few studies have focused on the effects of exposure to particulate matter and gaseous air pollutants on CHD. This study aimed to explore the relationship between air pollutants and the number of hospitalized patients with CHD in Lanzhou, and we collected daily data on the number of hospitalized patients with CHD, daily air pollutants, and meteorological factors from 2013 to 2020. A distributed lag nonlinear model (DLNM) combined with a quasi-Poisson regression model was applied to evaluate the relationship between air pollutants and the number of hospitalized patients with CHD. The results indicated that the hysteresis effect of all pollutants except O38h reached its maximum at lag3, and the relative risk of coronary heart disease admission was 1.0014 (95%CI: 1.0004, 1.0023), 1.0003 (95%CI: 1.0000, 1.0006), 1.0020 (95%CI: 1.0004, 1.0035), and 1.0053 (95%CI: 1.0026, 1.0080) when PM2.5, PM10, NO2, and SO2 concentrations were increased by 10 µg/m3, respectively. Each 1 mg/m3 increase in CO concentration was associated with a relative risk of coronary heart disease; hospitalization risk was 1.1076 (95%CI: 1.0530, 1.1650). We observed a relative risk of 0.9991 (95%CI: 0.9986, 0.9999) for each 10 µg/m3 increase in O38h for coronary heart disease admission at lag1. Women and elderly were more susceptible to the impact of air pollution, and the impact was greater during cold seasons. Our results indicate that air pollution increased the risk of hospitalization for CHD in a short term. The research findings can provide strategic insights into the impact of current and future air pollution on CHD.
Air Pollutants , Air Pollution , Coronary Disease , Humans , Female , Aged , Time Factors , Air Pollution/adverse effects , Air Pollutants/adverse effects , Hospitalization , Coronary Disease/epidemiology , China/epidemiology , Environmental Exposure/adverse effects
To understand the nutrient use strategies of 11 tree species in a subtropical common-garden, we measured the specific leaf area, nitrogen (N) and phosphorus (P) resorption and stoichiometric characteristics of leaves in August 2019. The results showed that the specific leaf area, N and P concentrations in mature and senescent leaves of evergreen broadleaved (Lindera communis, Cinnamomum camphora, Schima superba, Castanopsis carlesii, Michelia macclurei and Elaeocarpus decipiens) and coniferous species (Cunninghamia lanceolata and Pinus massoniana) were lower than those of deciduous broadleaved species (Liquidambar formosana, Sapindus mukorossi and Liriodendron chinense). In contrast, C:N and C:P in mature leaves of evergreen broadleaved and coniferous species were significantly higher than those of deciduous broadleaved species. Except for C. carlesii, the N:P of all the species were lower than 14. Compared with other tree species, N and P resorption efficiencies of S. mukorossi were higher than 50% based on both mass and leaf area. Although P resorption efficiency of P. massoniana, C. lanceolata and C. camphora were higher than 50%, N and P resorption efficiency of M. macclurei were the lowest with only 15%-30%. In addition, specific leaf area of mature leaves was significantly positively correlated with N and P concentrations, but negatively correlated with C:N and C:P. In the common-garden, evergreen broadleaved species such as C. carlesii and L. communis, and coniferous species such as P. massoniana might belong to the slow investment species with lower specific leaf area, N and P concentrations, displaying relatively efficient in N and P resorption and utilization in comparison with other species. In contrast, deciduous broadleaved species such as S. mukoraiensis might be the fast investment species with low N and P use efficiency. Interestingly, tree species being restricted by N availability did not exhibit higher N resorption efficiency in the common-garden. Similarly, C. carlesii, the only P-restricted species here, did not exhibit higher P resorption efficiency. Our results provided scientific support for afforestation practice in the mid-subtropics.
Cunninghamia , Trees , China , Nitrogen/analysis , Phosphorus , Plant Leaves/chemistry
OBJECTIVES: To investigate sex difference in global gastric cancer incidence by year, age and socioeconomical status. DESIGN: An international comparative study. SETTING: We obtained the global and national sex-specific incidence of gastric caner by year and age from the Global Burden of Disease Study 2017. The human development index (HDI) in 2017 as an indicator of national socioeconomical status was extracted from the Human Development Report. MAIN OUTCOME MEASURES: Sex-specific incidence of gastric cancer was compared by year and age at the global level. Linear regression analyses were performed to explore socioeconomic-associated sex difference in gastric cancer incidence. RESULTS: Despite declining incidence of global gastric cancer in both sexes between 1990 and 2017, relative sex difference showed an increasing trend, with male to female ratios of age-standardised incidence rates (ASRs) rising from 1.86 to 2.20. Sex difference was almost negligible under 45 years of age and relative difference maximised in the age range of 65-69 years with male to female ratios of ASRs being 2.74. Both absolute sex difference (standardised ß=0.256, p<0.001) and relative difference (standardised ß=0.387, p<0.001) in ASRs were positively associated with HDI. CONCLUSIONS: This study revealed that decreasing incidence of global gastric cancer was accompanied by widening sex difference in the past few decades. Men always had higher incidence than women. Greater sex difference was found in older age and in more developed countries. These findings highlight the importance of making sex-sensitive health policy to cope with the global gastric cancer burden.
Global Health , Stomach Neoplasms/epidemiology , Adolescent , Adult , Aged , Female , Gender Identity , Global Burden of Disease , Humans , Incidence , Male , Middle Aged , Sex Distribution , Sex Factors , Young Adult
INTRODUCTION: Gastrointestinal stromal tumors (GISTs), with a primary occurrence in the duodenum and proximal jejunum, are rare and treatment is poorly understood. This study aimed to evaluate the main factors influencing the prognosis of GIST resection in this complex anatomical structure. MATERIALS AND METHODS: This retrospective study included 47 patients who underwent surgery for primary GIST of the duodenum (20) and proximal jejunum (27) between 2012 and 2017. Perioperative clinical data as well as relapse and survival information were collected. RESULTS: All patients underwent negative margin resection (R0) of duodenal and proximal jejunum GISTs. Complications occurred more frequently in treatment of duodenal GISTs than proximal jejunum GISTs (pâ¯=â¯0.003). GISTs in D3 (the 3rd portion of duodenum) were related to larger tumor size (pâ¯=â¯0.001), higher probability of severe complication rate (pâ¯=â¯0.042), longer hospital stays (pâ¯=â¯0.023) and fasting time (pâ¯=â¯0.020). More complications were found for patients with digestive reconstruction than limited resection (pâ¯=â¯0.010). Additionally, patients with a tumor mass larger than 5â¯cm or a mitotic index greater than 5 mitoses/50 HPFs showed poorer therapeutic outcomes. The 1- and 3-year overall survival was 97.9% and 86.1%, respectively and were not influenced by operation type (pâ¯=â¯0.061) or GIST position (pâ¯=â¯0.447). CONCLUSION: With negative operational margins, limited resection is a safe and feasible procedure for duodenal and proximal jejunum GIST patients and unnecessary digestive reconstruction should be avoided. Considering the severe complication rate, resection for GISTs in D3 should be performed with care.
Digestive System Surgical Procedures/methods , Duodenal Neoplasms/surgery , Duodenum/pathology , Gastrointestinal Stromal Tumors/surgery , Jejunal Neoplasms/surgery , Jejunum/pathology , Neoplasm Staging/methods , Adult , Aged , Aged, 80 and over , China/epidemiology , Duodenal Neoplasms/diagnosis , Duodenum/surgery , Female , Follow-Up Studies , Gastrointestinal Stromal Tumors/diagnosis , Humans , Incidence , Jejunal Neoplasms/diagnosis , Jejunum/surgery , Male , Middle Aged , Postoperative Complications/epidemiology , Retrospective Studies , Survival Rate/trends , Young Adult
The Notch signaling pathway has been identified as a therapeutic target for cancers. γ-Secretase inhibitors (GSIs) have been progressively recognized as potential anticancer drugs. The present study aimed to investigate the effects of anti-delta like legend 4 (anti-DLL4) treatment on the anticancer efficacy of GSIs in gastric cancer. SGC-7901-GFP human gastric cancer cells were tested for DLL4 expression by rosette formation test and immunofluorescence, and then were treated with anti-DLL4 antibody N-[N-(3,5-difluorophenacetyl)-L-ananyl]-S-phenylglycine t-butyl ester (DAPT, a type of GSI), or a combination of anti-DLL4 antibody and DAPT. The effects of in vitro treatments on cell apoptosis, cell cycle, and cell invasion were analyzed. For in vivo study, an orthotopic mouse model of gastric cancer was established with green fluorescence expressing SGC-7901. Ultrasound targeted microbubble destruction was used to treat tumor-bearing mice with anti-DLL4 antibody conjugated microbubbles, DAPT, and a combination of the two. Real-time fluorescence imaging was performed to assess tumor cell inhibition in each group. Following in vivo treatments, apoptosis of tumor cells and the expression of apoptosis-related genes BAX, Bcl-2, and P53 were detected by TUNEL and immunohistochemical staining. In vivo combined treatment of anti-DLL4 and DAPT led to a higher rate of cell apoptosis and greater inhibition of cell invasion than that observed with DAPT treatment alone. DAPT and anti-DLL4 combination therapy resulted in decreased cell distribution at G1 phase and increased cell distribution at S phase, compared to the untreated control group (P < .01). In vivo combined therapy with anti-DLL4 and DAPT significantly increased tumor growth inhibition and tumor cell apoptosis when compared to DAPT therapy alone (P < .05). In addition, combined treatment significantly increased expression of BAX and P53 and reduced Bcl-2 expression (P < .05). Conversely, treatment with DAPT alone only increased expression of BAX and P53 (P < .05), suggesting that the reduction of Bcl-2 expression may play an important role in the synergetic antitumor and proapoptosis effects of the combined treatment. Concurrent treatment with anti-DLL4 enhances the antitumor and proapoptotic efficacy of the γ-secretase inhibitor in gastric cancer both in vitro and in vivo.
MicroRNA (miRNA) studies are experiencing a transition from basic research applications to clinical applications. However, the lack of reliable and sensitive miRNA detection methods has become a bottleneck in the process. Here, we report an absolute quantification method based on the competitive PCR amplification of specific miRNAs and synthetic RNA spike-ins in a single reaction. RNA spike-ins are quantified as dynamic RNA copy number standards and are used to measure selected miRNAs free from the effects of intra-assay variables, including those from individual sample sources. Combined with the size differentiation power of capillary electrophoresis, the content of miRNAs was reproducibly measured, with verifiable detection limits of 10-46 copies over 5-log detection ranges. The direct measurements of miRNAs from 168 human serum samples and their considerable value as a diagnostic for bronchopneumonia and bronchiolitis demonstrate the potential of the assay in clinical applications.
MicroRNAs/genetics , Polymorphism, Genetic , Electrophoresis, Capillary , Humans , Polymerase Chain Reaction , Polymorphism, Restriction Fragment Length , Reproducibility of Results
