DNA-based molecular classifiers for the profiling of gene expression signatures.

Although gene expression signatures offer tremendous potential in diseases diagnostic and prognostic, but massive gene expression signatures caused challenges for experimental detection and computational analysis in clinical setting. Here, we introduce a universal DNA-based molecular classifier for profiling gene expression signatures and generating immediate diagnostic outcomes. The molecular classifier begins with feature transformation, a modular and programmable strategy was used to capture relative relationships of low-concentration RNAs and convert them to general coding inputs. Then, competitive inhibition of the DNA catalytic reaction enables strict weight assignment for different inputs according to their importance, followed by summation, annihilation and reporting to accurately implement the mathematical model of the classifier. We validated the entire workflow by utilizing miRNA expression levels for the diagnosis of hepatocellular carcinoma (HCC) in clinical samples with an accuracy 85.7%. The results demonstrate the molecular classifier provides a universal solution to explore the correlation between gene expression patterns and disease diagnostics, monitoring, and prognosis, and supports personalized healthcare in primary care.

A PAM-free CRISPR/Cas12a ultra-specific activation mode based on toehold-mediated strand displacement and branch migration.

CRISPR (clustered regularly interspaced short palindromic repeats) technology has achieved great breakthroughs in terms of convenience and sensitivity; it is becoming the most promising molecular tool. However, only two CRISPR activation modes (single and double stranded) are available, and they have specificity and universality bottlenecks that limit the application of CRISPR technology in high-precision molecular recognition. Herein, we proposed a novel CRISPR/Cas12a unrestricted activation mode to greatly improve its performance. The new mode totally eliminates the need for a protospacer adjacent motif and accurately activates Cas12a through toehold-mediated strand displacement and branch migration, which is highly universal and ultra-specific. With this mode, we discriminated all mismatch types and detected the EGFR T790M and L858R mutations in very low abundance. Taken together, our activation mode is deeply incorporated with DNA nanotechnology and extensively broadens the application boundaries of CRISPR technology in biomedical and molecular reaction networks.

RART-LAMP: One-Step Extraction-Free Method for Genotyping within 40 min.

Loop-mediated isothermal amplification (LAMP) is a commonly used alternative to PCR for point-of-care detection of nucleic acids due to its rapidity, sensitivity, specificity, and simpler instrumentation. While dual-labeled TaqMan probes are widely used in PCR for single-nucleotide polymorphism (SNP) genotyping, real-time LAMP primarily relies on turbidimetry or intercalator fluorescence measurements, which can be non-specific and generate false-positive results. In this study, we propose a closed-tube, dual-labeled RNA-modified probes and RNase H II-assisted real-time LAMP (RART-LAMP) method for SNP genotyping. Our findings indicate that (1) fluorescence signals were predominantly derived from probe hydrolysis rather than hybridization, (2) temperature-controlled hybridization between the probe and template ensured the specificity of SNP analysis, and (3) RNase H II hydrolysis between the target containing SNP sites and probes did not exhibit sequence specificity. Our RART-LAMP approach demonstrated excellent performance in genotyping C677T clinical samples, including gDNA extracted from blood, saliva, and swabs. More importantly, saliva and swab samples could be directly analyzed without any pretreatment, indicating promising prospects for nucleic acid analysis at the point of care in resource-limited settings.

Identification of genes related to low-grade glioma progression and prognosis based on integrated transcriptome analysis.

Glioma is one of the most common types of human brain tumor, with high mortality in high-grade gliomas (HGG). Low-grade gliomas (LGG) can progress into HGG, leading to poor prognosis. However, it is unclear what factors affect the progression of LGG to HGG. This study aims to explore the function of the crosstalk genes on the progression and prognosis of LGG using bioinformatics analysis. Integrated transcriptome analysis was used to screen differentially expressed genes (DEGs). Then, gene ontology (GO) function enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed to investigate the association between DEGs and gene functions and pathways by ClusterProfiler package and ClueGO plug-in. Protein-protein interaction (PPI) network analysis was applied to explore the connection between genes and biological processes. Subsequently, the gene clusters were analyzed using the Centiscape and molecular complex detection (MCODE) plug-in in Cytoscape software, where the crosstalk genes were identified for further study. Ultimately, the UALCAN website and Gene Expression Profiling Interactive Analysis (GEPIA) website were performed to visualize the expression levels and survival curves of genes, respectively. There were 74 DEGs identified in glioma, including 55 upregulated genes and 19 downregulated genes, which mainly were enriched in extracellular matrix (ECM)-receptor interaction, focal adhesion, PI3K-Akt signaling pathway, and so on. Then, six crosstalk genes were selected, including COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, and COL5A2 genes. Overall survival (OS) analysis of crosstalk genes was conducted on the GEPIA website. High expression levels of crosstalk genes were closely related to the low survival rate of patients with LGG. The overexpressed crosstalk genes, such as COL1A1, COL1A2, COL3A1, COL4A1, COL4A2, and COL5A2 may participate in the progression and poor prognosis of LGG through the ECM-receptor interaction pathway.

Phage-Guided Targeting, Discriminative Imaging, and Synergistic Killing of Bacteria by AIE Bioconjugates.

New agents with particular specificity toward targeted bacteria and superefficacy in antibacterial activity are urgently needed in facing the crisis of worldwide antibiotic resistance. Herein, a novel strategy by equipping bacteriophage (PAP) with photodynamic inactivation (PDI)-active AIEgens (luminogens with aggregation-induced emission property) was presented to generate a type of AIE-PAP bioconjugate with superior capability for both targeted imaging and synergistic killing of certain species of bacteria. The targeting ability inherited from the bacteriophage enabled the bioconjugates to specifically recognize the host bacteria with preserved infection activity of phage itself. Meanwhile, the AIE characteristic empowered them a monitoring functionality, and the real-time tracking of their interactions with targets was therefore realized via convenient fluorescence imaging. More importantly, the PDI-active AIEgens could serve as powerful in situ photosensitizers producing high-efficiency reactive oxygen species (ROS) under white light irradiation. As a result, selective targeting and synergistic killing of both antibiotic-sensitive and multi-drug-resistant (MDR) bacteria were successfully achieved in in vitro and in vivo antibacterial tests with excellent biocompatibility. This novel AIE-phage integrated strategy would diversify the existing pool of antibacterial agents and inspire the development of promising drug candidates in the future.

Analysis of drug-resistance-associated mutations and genetic barriers in hepatitis C virus NS5B sequences in China.

The hepatitis C virus (HCV) NS5B protein is an RNA-dependent RNA polymerase that is required for viral genome replication and constitutes the most important target region for drugs being developed as direct-acting antivirals (DAAs) against HCV genotype 1. However, the extreme genetic variability leading to drug resistance mutations and genetic barriers has dramatically compromised the effectiveness of DAA therapy. The purpose of this study was to analyze the genetic variability of NS5B polymerase in HCV patients from different provinces of China to identify the impact of these resistance sites on genetic barriers. We analyzed 3489 NS5B sequences of HCV strains circulating in different regions of China, obtained from the GenBank database, 153 of which were from three cities in Sichuan Province (Yibin, Zigong and Zhangzhou). Sequence alignment was conducted using MEGA 6.0, the genetic information was translated into amino acids, and the percentage of polymorphic amino acid sites was calculated. The Vijver method was used to evaluate the occurrence of genetic barriers in HCV NS5B sequences. Blood samples were collected from 153 HCV patients from Sichuan for NS5B sequence analysis using real-time PCR and the Sanger method. Of the 17 antiviral drug resistance sites summarized from the published literature, nine were found in Chinese NS5B sequences, and C316Y was identified as the dominant mutation. Analysis of genetic barriers revealed that the probability of mutation to a drug-resistance-associated amino acid, in response to selective pressure from antiviral drugs was 100% at site 96 and 99.7% at site 282. Our study is the first to analyze the drug resistance sites and to evaluate genetic barriers in NS5B sequences that could affect the responsiveness of Chinese HCV patients to DAA therapy. The results provide a valuable basis for drug development and introduction of foreign-origin antiviral drugs in China that targeting the HCV NS5B region.

A fluorometric assay for rapid enrichment and determination of bacteria by using zirconium-metal organic frameworks as both capture surface and signal amplification tag.

A fluorometric assay was introduced to determine Acinetobacter baumannii (A. baumannii) in blood samples by utilizing Zr-MOFs both as functional coating for magnetic Fe3O4 nanoparticles to provide modification surface (Zr-mMOF) and as fluorescein carrier to produce fluorescence signals (F@UIO-66-NH2). Through strong Zr-O-P bonding, two distinct terminal phosphate-labeled A. baumannii and lipopolysaccharide (LPS) specific aptamers were attached onto Zr-MOFs to fabricate the magnetic core-shell capture probe (denoted as Zr-mMOF-p-Ab-Apt) and signal probe (denoted as F@UIO-66-NH2-p-LPS-Apt), respectively. After successive incubation with A. baumannii in blood samples and magnetic separation, the sandwich-type composite of capture probe/A. baumannii cells/signal probe was treated with high concentration of anionic phosphate ions to destroy the nano-structure of UIO-66-NH2 in the signal probe and fast release of fluorescein to produce amplified fluorescence signals. Due to the high aptamer modification efficiency of Zr-mMOF-p-Ab-Apt (up to 93%) and its strong affinity to A. baumannii, the enrichment efficiency of this capture probe has reached to 96.7%. Further, due to the high fluorescein loading efficiency of UIO-66-NH2 and our novel amplification strategy to destroy F@UIO-66-NH2-p-LPS-Apt to release and amplify fluorescein signals at 512 nm in the presence of high concentration of anionic phosphate ions, the sensitivity of this method has reached 10 cfu mL-1. This method allows enrichment and determination of A. baumannii within ~2.5 h. The limit of detection of A. baumannii in blood samples is 10 cfu mL-1 with a linear range of 101-105 cfu mL-1. This indicates the potential of this assay for diagnosis of bloodstream infection in early stage. Graphical abstractSchematic representation of sandwich-type fluorometric assay for Acinetobacter baumannii in blood samples with the capture probe (Zr-mMOF-p-Ab-Apt) and signal probe (F@UIO-66-NH2-p-LPS-Apt). The limit of detection is down to 10 cfu mL-1 with a linear range of 101-105 cfu mL-1.

Simultaneous colorimetric determination of acute myocardial infarction biomarkers by integrating self-assembled 3D gold nanovesicles into a multiple immunosorbent assay.

An improved enzyme-free immunosorbent assay is described for the simultaneous detection of the myocardial infarction biomarkers N-terminal pro B type natriuretic peptide (NT-proBNP), creatine kinase-MB (CK-MB), and cardiac muscle troponin T (cTnT). The assay integrates 3D gold nanovesicles (GNVs) and three allochroic agents (phenolphthalein, methyl red, bromothymol blue). The pH regulated allochroic agents were enwrapped in GNVs to acts as ultrasensitive nanoprobes. Loading can be controlled by adjusting the temperature to efficiently load and release the allochroic agents. This bare-eye multicolor assay has limits of detection of 70 pg·mL-1 for NT-proBNP, 910 pg·mL-1 for CK-MB, and 7.8 pg·mL-1 for cTnT. Other features include (a) a linear range that extends over a wide range and sometimes is better than conventional HRP-based immunoassays, and (b) a precision that is comparable to immunofluorescence assays as used in the clinical laboratory. Graphical abstract Schematic presentation of an improved enzyme-free immunosorbent assay (EFISA). It integrates 3D gold nano-vesicles (GNVs) and allochroic agents for the simultaneous detection of acute myocardial infarction (AMI) biomarkers (N-terminal prohormone of brain natriuretic peptide (NT-proBNP), kinase-muscle/brain test (CK-MB), and cardiac muscle troponin (cTnT)).

Fluorometric determination of microRNA by using an entropy-driven three-dimensional DNA walking machine based on a catalytic hairpin assembly reaction on polystyrene microspheres.

An entropy-driven 3-D DNA walking machine is presented which involves catalytic hairpin assembly (CHA) for detection of microRNA. A 3-D DNA walking machine was designed that uses streptavidin-coated polystyrene microspheres as track carriers to obtain reproducibility. The method was applied to microRNA 21 as a model analyte. Continuous walking on the DNA tracks is achieved via entropy increase. This results in a disassembly of ternary DNA substrates on polystyrene microspheres and leads to cycling of microRNA 21. The release of massive auxiliary strands from ternary DNA substrates induces the CHA. This is accompanied by in increase in fluorescence, best measured at excitation/emission wavelengths of 480/520 nm. On account of entropy-driven reaction, the assay is remarkably selective. It can differentiate microRNA 21 from homologous microRNAs in giving a signal that is less than 5% of the signal for microRNA 21 except for microRNA-200b. The assay works in the 50 pM to 20 nM concentration range and has a 41 pM detection limit. The method displays good reproducibility (between 1.1 and 4.2%) and recovery (from 99.8 to 104.0%). Graphical abstract An entropy-driven 3-D DNA walking machine is described. It is based on the use of polystyrene microspheres and of a catalytic hairpin assembly reaction for sensitive microRNA detection. Figure Notes: AS represents auxiliary strand; S represents substrate strand; LS represents link strand; F represents fuel nucleic acid; RepF represents nucleic acid labeled with FAM; RepQ represents nucleic acid labeled with BHQ1.

A target-triggered biosensing platform for detection of HBV DNA based on DNA walker and CHA.

Hepatitis B virus (HBV), one of the causative agents of viral hepatitis, may lead to chronic hepatitis, cirrhosis, and liver cancer. In this work, we designed a sensitive and modular biosensing platform for detecting HBV DNA based on a DNA walker that hangs on to surfaces and a catalyst-triggered catalyzed hairpin assembly (CHA). In the presence of HBV DNA, strand displacement reaction between target and double-stranded complex caused the release of walker strand to trigger the DNA walker. Then, a catalyst was free to open the trapped hairpins to form a new double-strand complex, driving the CHA reaction. Thus, a powerful cascade amplification reaction realized in DNA walker and CHA based on toehold-mediated strand displacement reaction in this system. To achieve quantitative detection of HBV DNA, a fluorescent-quencher signaling pair was employed, the turn-on fluorescence provided an analytical signal. A wide detection range from 0.5 nM to 50 nM with a detection limit as low as 0.20 nM was reached on the condition of acceptable specificity and reproducibility. We could also further apply it to multiple different bioanalysis by changing adjustable elements. This reported biosensor opened a new avenue for sensitivity and modularity of DNA detection.

An enzyme free electrochemical biosensor for sensitive detection of miRNA with a high discrimination factor by coupling the strand displacement reaction and catalytic hairpin assembly recycling.

An isothermal, enzyme free, ultra-specific and ultra-sensitive protocol for electrochemical detection of miRNAs is proposed based on the toehold-mediated strand displacement reaction (SDR) and non-enzymatic catalytic hairpin reaction (CHA) recycling. The SDR was first triggered only in the presence of target miRNA and this process also affects other miRNA interferences having similar target sequences, thus guaranteeing a high discrimination factor and could be used in rare content miRNA detection with various amounts of interferences having similar target sequences. The output protector strand then triggered enzyme free CHA amplification and generates plenty of hairpin self-assembly products. This process in turn influences SDR equilibrium to move to the right and generates large amounts of protector output to ensure analysis sensitivity. Compared with traditional CHA, our proposed method greatly improved the signal to noise ratio and shows excellent performance in rare miRNA detection with miRNA analogue interference. Under the optimal experimental conditions and using square wave voltammetry, the established biosensor could detect target miRNA-21 down to 30 fM (S/N = 3) with a dynamic range from 100 fM to 2 nM, and discriminate rare target miRNA-21 from mismatched miRNA with high selectivity. This method holds great promise in miRNA detection from human cancer cell lines and would be a versatile and powerful tool for clinical molecular diagnostics.

The interplay between the tumor microenvironment and tumor-derived small extracellular vesicles in cancer development and therapeutic response.

The tumor microenvironment (TME) plays an essential role in tumor cell survival by profoundly influencing their proliferation, metastasis, immune evasion, and resistance to treatment. Extracellular vesicles (EVs) are small particles released by all cell types and often reflect the state of their parental cells and modulate other cells' functions through the various cargo they transport. Tumor-derived small EVs (TDSEVs) can transport specific proteins, nucleic acids and lipids tailored to propagate tumor signals and establish a favorable TME. Thus, the TME's biological characteristics can affect TDSEV heterogeneity, and this interplay can amplify tumor growth, dissemination, and resistance to therapy. This review discusses the interplay between TME and TDSEVs based on their biological characteristics and summarizes strategies for targeting cancer cells. Additionally, it reviews the current issues and challenges in this field to offer fresh insights into comprehending tumor development mechanisms and exploring innovative clinical applications.

Magnetically modified bacteriophage-triggered ATP release activated EXPAR-CRISPR/Cas14a system for visual detection of Burkholderia pseudomallei.

Burkholderia pseudomallei, widely distributed in tropical and subtropical ecosystems, is capable of causing the fatal zoonotic disease melioidosis and exhibiting a global trend of dissemination. Rapid and sensitive detection of B. pseudomallei is essential for environmental monitoring as well as infection control. Here, we developed an innovative biosensor for quantitatively detecting B. pseudomallei relies on ATP released triggered by bacteriophage-induced bacteria lysis. The lytic bacteriophage vB_BpP_HN01, with high specificity, is employed alongside magnetic nanoparticles assembly to create a biological receptor, facilitating the capture and enrichment of viable target bacteria. Following a brief extraction and incubation process, the captured target undergoes rapid lysis to release contents including ATP. The EXPAR-CRISPR cascade reaction provides an efficient signal transduction and dual amplification module that allowing the generated ATP to guide the signal output as an activator, ultimately converting the target bacterial amount into a detectable fluorescence signal. The proposed bacteriophage affinity strategy exhibited superior performance for B. pseudomallei detection with a dynamic range from 10^2 to 10^7 CFU mL-1, and a LOD of 45 CFU mL-1 within 80 min. Moreover, with the output signal compatible across various monitoring methods, this work offers a robust assurance for rapid diagnosis and on-site environmental monitoring of B. pseudomallei.

​Meta-analysis of the applied value of the growth differentiation factor 15 detection in HFpEF diagnosis.

OBJECTIVE: To systematically evaluate the diagnostic value of growth differentiation factor-15 (GDF-15) for heart failure with preserved ejection fraction (HFpEF). METHODS: Chinese and English literature on the diagnosis of HFpEF using GDF-15 were searched in PubMed, Embase, Web of Science (WOS), Cochrane Library, China National Knowledge Infrastructure (CNKI), China Science and Technology Journal Database, WanFang Database, and others. The literature on the diagnostic value of the GDF-15 test for HFpEF was screened from the establishment of the database to April 2023 according to the inclusion and exclusion criteria. The quality of the included studies was then assessed based on the QUADAS-2 list, and the threshold effect was evaluated using the Meta-Disc1.4 software. STATA 17.0 software was used to combine the sensitivity, specificity, and area under the curve (AUC) of the included studies. Moreover, heterogeneity was evaluated by the inconsistency index (I2) and Cochrane Q index, and the source of heterogeneity was explored by subgroup analysis, meta-regression, and sensitivity analysis. Finally, Deek's quantitative funnel plot was used to assess whether there was publication bias among the included studies. RESULTS: A total of ten studies involving 1550 patients were included. The pooled sensitivity was 0.77 (95%CI: 0.70-0.83), the specificity was 0.79 (95%CI: 0.68-0.87), the positive likelihood ratio was 3.9 (95%CI: 2.6-5.9), and the negative likelihood ratio was 0.21 (95%CI:0.12-0.36). The diagnostic odds ratio was 19 (95%CI: 9-37), and the AUC of SROC was 0.88 (95%CI: 0.85-0.9). The results of the heterogeneity test showed significant heterogeneity among the studies (I2 = 96%, p = 0.000 < 0.01). Meta-regression analysis showed that there was a significant difference in diagnostic efficacy between the gold standard group (p = 0.0064 < 0.05), while there was no significant difference in diagnostic efficacy among the three subgroups of age, gender, and comprehensive group (p > 0.05). After excluding the articles that did not include biomarkers for the diagnosis of HFpEF, the average age ≥73 years old, and the proportion of women >55%, the remaining four articles had the pooled sensitivity of 0.80 (I2 = 60.1%, p = 0.06 >​ 0.05) and the pooled specificity of 0.84 (I2 = 0%, p = 0.61 >​0.05), which insisted that there is no significant heterogeneity among them. CONCLUSION: With its high sensitivity and specificity for HFpEF diagnosis, GDF-15 is a novel biomarker for HFpEF diagnosis.

A hairpin probe-mediated exponential amplification reaction for highly sensitive and specific detection of microRNAs.

In this work, we propose a hairpin probe-mediated exponential amplification reaction (HEAR) strategy that combines DNA strand displacement with a "who triggers, who gets generated" mode, providing excellent single-base discrimination and a reduced background signal. The detection limit is 19 aM, which is reduced by 3 orders of magnitude compared to traditional exponential amplification approaches. This one-pot strategy also exhibits a wide dynamic range, high specificity and short detection time. It is expected to become a powerful tool for clinical diagnosis.

Blocker displacement amplification mediated PCR based screen-printed carbon electrode biosensor and lateral flow strip strategy for CYP2C19*2 genotyping.

Single nucleotide variants in CYP2C19*2 are associated with clopidogrel resistance in coronary heart disease. In order the guidance the dosage of drug and personalized medicine, blocker displacement amplification was first used to specific amplify G site and A site alleles. For electrochemical strategy, forward primers were labeled electrochemical active methyl blue and ferrocene, generates signals on -0.26 for G site and 0.22 V for A site. For lateral flow strip assay, primers with specific modification were used to generates unique color in test line 1 for G site and test line 2 for A site. In conclusion, we developed a sensitive screen-printed carbon electrodes based electrochemical sensor and gold nano particle based lateral flow strip assay strategy to successfully genotyping CYP2C19*2 GG, GA and AA genotype. The proposed method can realize CYP2C19*2 analysis from multiple biological samples including whole blood, buccal swab, saliva and hair root, and showed good consistency with Sequencing. Due to the fact our proposed strategy merely relies on thermal cycler instrument and visual strip detection, this platform shows great potential in source-limited regions genotyping.

Ethanol promoting the upregulation of C-X-C Motif Chemokine Ligand 1（CXCL1） and C-X-C Motif Chemokine Ligand 6（CXCL6） in models of early alcoholic liver disease.

Alcoholic liver disease (ALD) denotes a series of liver diseases caused by ethanol. Recently, immune-related genes (IRGs) play increasingly crucial role in diseases. However, it's unclear the role of IRGs in ALD. Bioinformatic analysis was used to discern the core immune-related differential genes (IRDGs) in the present study. Subsequently, Cell Counting Kit-8 say, oil red O staining, and triglyceride detection were employed to explore optimal experimental conditions of establishing hepatocellular models of early ALD. Ultimately, real-time reverse transcription-PCR and immunohistochemistry/immunocytochemistry methods were adopted to verify the expressions of mRNA and proteins of core IRDGs, respectively. C-X-C Motif Chemokine Ligand 1 (Cxcl1) and Cxcl6 were regarded as core IRDGs via integrated bioinformatics analysis. Besides, Lieber Decarli Ethanol feeding and 200 mM and 300 mM ethanol stimulating L02 cells for 36 h can both successfully hepatocellular model. In ethanol groups, the levels of CXCL1 and CXCL6 mRNA were significantly upregulated than pair-fed groups (P < 0.0001). Also, immunohistochemistry revealed that positive particles of CXCL1 and CXCL6 in mice model of early ALD were obviously more than control groups (P < 0.0001). Besides, in L02 hepatocytes stimulated by ethanol, CXCL1 and CXCL6 mRNA were over-expressed, compared with normal L02 cells (P < 0.0001). Meanwhile, immunocytochemistry indicated that CXCL1 and CXCL6 proteins in hepatocellular model of early ALD were higher than normal L02 hepatocytes stimulus (P < 0.0001). Ethanol promoted the upregulation of Cxcl1 and Cxcl6 mRNA and proteins in models of early ALD, denoting their potentiality of acting as biomarkers of ALD.

Specific and robust hybridization based on double-stranded nucleic acids with single-base resolution.

Nucleic acid hybridization plays a critical role in medical diagnostics and nanotechnology, but its selectivity and robustness remain to be improved. Here, focusing on double-stranded nucleic acid-based hybridization, we present a series of related strategies. Above all, two simple strategies for enriching toehold-included double-stranded nucleic acids have been proposed. On this basis, two universal hybridization methods with higher selectivity than typical toehold exchange reaction and a long target detection method using short probes to extend the detectable length range are realized. We also provide a double-stranded nucleic acids-catalyzed cycle amplification reaction to improve sensitivity, which has superior interference resistance and excellent discrimination for single-base mismatches. Besides, double-stranded nucleic acids with forked toeholds are used as essential elements to construct a series of logic gates that can evaluate different input combinations. Given the unique advantages of double-stranded nucleic acids, we expect the current work to advance the application of double-stranded nucleic acid-based hybridization in medical diagnostics and nanotechnology.

Cooperative Branch Migration: A Mechanism for Flexible Control of DNA Strand Displacement.

DNA strand displacement plays an essential role in the field of dynamic DNA nanotechnology. However, flexible regulation of strand displacement remains a significant challenge. Most previous regulatory tools focused on controllable activation of toehold and thus limited the design flexibility. Here, we introduce a regulatory tool termed cooperative branch migration (CBM), through which DNA strand displacement can be controlled by regulating the complementarity of branch migration domains. CBM shows perfect compatibility with the majority of existing regulatory tools, and when combined with forked toehold, it permits continuous fine-tuning of the strand displacement rate spanning 5 orders of magnitude. CBM manifests multifunctional regulation ability, including rate fine-tuning, continuous dynamic regulation, reaction resetting, and selective activation. To exemplify the powerful function, we also constructed a nested if-function signal processing system on the basis of cascading CBM reactions. We believe that the proposed regulatory strategy would effectively enrich the DNA strand displacement toolbox and ultimately promote the construction of DNA machines of higher complexity in nucleic acid research and biomedical applications.

Exploration of a Robust and Prognostic Immune Related Gene Signature for Cervical Squamous Cell Carcinoma.

Background: Cervical squamous cell carcinoma (CESC) is one of the most frequent malignancies in women worldwide. The level of immune cell infiltration and immune-related genes (IRGs) can significantly affect the prognosis and immunotherapy of CESC patients. Thus, this study aimed to identify an immune-related prognostic signature for CESC. Methods: TCGA-CESC cohorts, obtained from TCGA database, were divided into the training group and testing group; while GSE44001 dataset from GEO database was viewed as external validation group. ESTIMATE algorithm was applied to evaluate the infiltration levels of immune cells of CESC patients. IRGs were screened out through weighted gene co-expression network analysis (WGCNA). A multi-gene prognostic signature based on IRGs was constructed using LASSO penalized Cox proportional hazards regression, which was validated through Kaplan-Meier, Cox, and receiver operating characteristic curve (ROC) analyses. The abundance of immune cells was calculated using ssGSEA algorithm in the ImmuCellAI database, and the response to immunotherapy was evaluated using immunophenoscore (IPS) analysis and the TIDE algorithm. Results: In TCGA-CESC cohorts, higher levels of immune cell infiltration were closely associated with better prognoses. Moreover, a prognostic signature was constructed using three IRGs. Based on this given signature, Kaplan-Meier analysis suggested the significant differences in overall survival (OS) and the ROC analysis demonstrated its robust predictive potential for CESC prognosis, further confirmed by internal and external validation. Additionally, multivariate Cox analysis revealed that the three IRGs signature served as an independent prognostic factor for CESC. In the three-IRGs signature low-risk group, the infiltrating immune cells (B cells, CD4/8 + T cells, cytotoxic T cells, macrophages and so on) were much more abundant than that in high-risk group. Ultimately, IPS and TIDE analyses showed that low-risk CESC patients appeared to present with a better response to immunotherapy and a better prognosis than high-risk patients. Conclusion: The present prognostic signature based on three IRGs (CD3E, CD3D, LCK) was not only reliable for survival prediction but efficient to predict the clinical response to immunotherapy for CESC patients, which might assist in guiding more precise individual treatment in the future.