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Abstract The precise regulation of cellular behaviors within a confined, crowded intracellular environment is highly amenable in diagnostics and therapeutics. While synthetic circuitry system through a concatenated chemical reaction network has rarely been reported to mimic dynamic self‐assembly system. Herein, a catalytic self‐defined circuit (CSC) for the hierarchically concatenated assembly of DNA domino nanostructures is engineered. By incorporating pre‐sealed symmetrical fragments into the preying hairpin reactants, the CSC system allows the hierarchical DNA self‐assembly via a microRNA (miRNA)‐powered self‐sorting catalytic hybridization reaction. With minimal strand complexity, this self‐sustainable CSC system streamlined the circuit component and achieved localization‐intensified cascaded signal amplification. Profiting from the self‐adaptively concatenated hybridization reaction, a reliable and robust method has been achieved for discriminating carcinoma tissues from the corresponding para‐carcinoma tissues. The CSC‐sustained self‐assembly strategy provides a comprehensive and smart toolbox for organizing various hierarchical DNA nanostructures, which may facilitate more insights for clinical diagnosis and therapeutic assessment.

BackgroundThe relationship between hormonal fluctuations in the reproductive system and the occurrence of low back pain (LBP) has been widely observed. However, the causal impact of specific variables that may be indicative of hormonal and reproductive factors, such as age at menopause (ANM), age at menarche (AAM), length of menstrual cycle (LMC), age at first birth (AFB), age at last live birth (ALB) and age first had sexual intercourse (AFS) on low back pain remains unclear.MethodsThis study employed Bidirectional Mendelian randomization (MR) using publicly available summary statistics from Genome Wide Association Studies (GWAS) and FinnGen Consortium to investigate the causal links between hormonal and reproductive factors on LBP. Various MR methodologies, including inverse-variance weighted (IVW), MR-Egger regression, and weighted median, were utilized. Sensitivity analysis was conducted to ensure the robustness and validity of the findings. Subsequently, Multivariate Mendelian randomization (MVMR) was employed to assess the direct causal impact of reproductive and hormone factors on the risk of LBP.ResultsAfter implementing the Bonferroni correction and conducting rigorous quality control, the results from MR indicated a noteworthy association between a decreased risk of LBP and AAM (OR=0.784, 95% CI: 0.689-0.891; p=3.53E-04), AFB (OR=0.558, 95% CI: 0.436-0.715; p=8.97E-06), ALB (OR=0.396, 95% CI: 0.226-0.692; p=0.002), and AFS (OR=0.602, 95% CI: 0.518-0.700; p=3.47E-10). Moreover, in the reverse MR analysis, we observed no significant causal effects of LBP on ANM, AAM, LMC and AFS. MVMR analysis demonstrated the continued significance of the causal effect of AFB on LBP after adjusting for BMI.ConclusionOur study explored the causal relationship between ANM, AAM, LMC, AFB, AFS, ALB and the prevalence of LBP. We found that early menarche, early age at first birth, early age at last live birth and early age first had sexual intercourse may decrease the risk of LBP. These insights enhance our understanding of LBP risk factors, offering valuable guidance for screening, prevention, and treatment strategies for at-risk women.

Background: Heterotopic ossification of tendons and ligaments (HOTL) is a common clinical condition characterized by the absence of discernible features and a lack of effective treatment. In vitro experiments have demonstrated that mechanical stimulation can induce cell differentiation toward osteogenesis, thereby promoting heterotopic ossification. Currently, there are few experimental designs aimed at inducing ligament stretching in mice, and the mechanism of heterotopic ossification may not entirely mirror that observed in clinical cases. Therefore, there is an urgent imperative to develop a novel and feasible animal model.Methods: In this study, all the Enpp1 gene deficiency mice (a mouse model with heterotopic ossification of multiple ligaments) were divided into three groups: the control group, the spinal brake group, and the hyperactive group (treadmill training group). An external spinal fixation device was designed to restrict mice’s spinal flexion and extension at 6 weeks of age. The brace was adjusted weekly according to the changes in the size of the mice. Additionally, treadmill training was used to increase activity in the spinal ligaments and Achilles tendons of the mice. Micro-CT scanning and HE staining were performed at 12, 20, and 28 W to evaluate the degree of ossification in the spinal ligament and Achilles tendon. What’s more, As one of the mechanical stimulation transduction signals, YAP plays a crucial role in promoting osteogenic differentiation of cells. Immunofluorescence was utilized to assess YAP expression levels for the purpose of determining the extent of mechanical stimulation in tissues.Results: Our findings showed that a few ossification lesions were detected behind the vertebral space of mice at 8 weeks of age. Spinal immobilization effectively restricts the flexion and extension of cervical and thoracic vertebrae in mice, delaying spinal ligament ossification and reducing chronic secondary spinal cord injury. Running exercises not only enhance the ossification area of the posterior longitudinal ligament (PLL) and Achilles tendons but also exacerbate secondary spinal cord injury. Further immunofluorescence results revealed a notable increase in YAP expression levels in tissues with severe ossification, suggesting that these tissues may be subjected to higher mechanical stimulation.Conclusion: Mechanical stimulation plays a pivotal role in the process of heterotopic ossification in tissues. Our study provided valid animal models to further explore the pathological mechanism of mechanical stimulation in HOTL development.

Abstract Probing endogenous molecules in living entities is significant to help decipher biological functions and exploit novel theranostics. DNA circuits that can recognize molecular inputs of interest and transduce them into readable signal outputs in an isothermal and autonomous manner have been actively pursued as versatile toolkits for intracellular biosensing research. Tremendous efforts are being devoted to developing integrated DNA circuits with high sensitivity, while spatiotemporal selectivity is often overlooked in the construction of functional DNA circuitry systems. This requires the development of stimuli‐responsive DNA circuits that can be activated on‐demand from the initial sensing‐blunt state to the sensing‐ready state under a programmable manner, for achieving precise bioimaging with high spatiotemporal control. In this review, an overview of recent advances in the construction of stimuli‐responsive DNA circuits that respond to particular triggers, including external physical stimuli and endogenous biological cues, and their spatiotemporally controllable molecular bioimaging applications is provided. The current challenges and potential solutions of these stimuli‐responsive DNA circuits for their future developments in this emerging field are also discussed.

Background: Intervertebral disc degeneration (IDD) is the leading cause of lower back pain, and an overall understanding of the molecular mechanisms related to IDD is still lacking. The purpose of this study was to explore gene signatures and immune cell infiltration related to IDD via bioinformatics analysis.Methods: A total of five expression profiles of mRNA and non-coding RNA were downloaded from the Gene Expression Omnibus (GEO) database. The potentially involved lncRNA/circRNA–miRNA–mRNA networks and protein-protein interaction networks were constructed by miRNet, circBank, STRING, and the Cytoscape database. Gene ontology, Kyoto Encyclopaedia of Genes and Genomes Analysis, Gene Set Enrichment Analysis, Gene Set Variation Analysis, Immune Infiltration Analysis, and Drug-Gene Interaction were used to analyse the top 20 hub genes. RT-qPCR was conducted to confirm the 12 differential expressions of genes both in the nucleus pulposus and annulus fibrosus tissuesResults: There were 346 differentially expressed mRNAs, 12 differentially expressed miRNAs, 883 differentially expressed lncRNAs, and 916 differentially expressed circRNAs in the GEO database. Functional and enrichment analyses revealed hub genes associated with platelet activation, immune responses, focal adhesion, and PI3K-Akt signalling. The apoptotic pathway, the reactive oxygen species pathway, and oxidative phosphorylation play an essential role in IDD. Immune infiltration analysis demonstrated that the Treg cells had significant infiltration, and three levels of immune cells, including dendritic cells, Th2 cells, and tumour-infiltrating lymphocytes, were inhibited in IDD. Drug-gene interaction analysis showed that COL1A1 and COL1A2 were targeted by collagenase clostridium histolyticum, ocriplasmin, and PDGFRA was targeted by 66 drugs or molecular compounds. Finally, 24 cases of IDD tissues and 12 cases of normal disc tissues were collected, and the results of RT-qPCR were consistent with the bioinformatics results.Conclusion: Our data indicated that the 20 hub genes and immune cell infiltration were involved in the pathological process of IDD. In addition, the PDGFRA and two potential drugs were found to be significant in IDD development.

Abstract Background Ossification of the posterior longitudinal ligaments (OPLL) is common disorder characterized by heterotopic ossification of the spinal ligaments. Mechanical stimulation (MS) plays an important role in OPLL. DLX5 is an essential transcription factor required for osteoblast differentiation. However, the role of DLX5 during in OPLL is unclear. This study aims to investigate whether DLX5 is associated with OPLL progression under MS. Methods Stretch stimulation was applied to spinal ligaments cells derived from OPLL (OPLL cells) and non‐OPLL (non‐OPLL cells) patients. Expression of DLX5 and osteogenesis‐related genes were determined by quantitative real‐time polymerase chain reaction and Western blot. The osteogenic differentiation ability of the cells was measured using alkaline phosphatase (ALP) staining and alizarin red staining. The protein expression of DLX5 in the tissues and the nuclear translocation of NOTCH intracellular domain (NICD) was examined by immunofluorescence. Results Compared with non‐OPLL cells, OPLL cells expressed higher levels of DLX5 in vitro and vivo (p

Blood always shows some immune changes after spinal cord injury (SCI), and detection of such changes in blood may be helpful for diagnosis and treatment of SCI. However, studies to date on blood immune changes after SCI in humans are not comprehensive. Therefore, to obtain the characteristics of blood immune changes and immunodiagnostic blood biomarkers of SCI and its different grades, a human blood transcriptome sequencing dataset was downloaded and analyzed to obtain differentially expressed immune-related genes (DEIGs), related functions and signaling pathways related to SCI and its various grades. Characteristic biomarkers of SCI and its different grades were identified by using weighted gene coexpression network analysis (WGCNA) and least absolute shrinkage and selection operator (LASSO) logistic regression. Expression of biomarkers was verified through experiments. The area under the curve (AUC) of biomarkers was calculated to evaluate their diagnostic value, and differences in immune cell content were examined. In this study, 17 kinds of immune cells with different contents between the SCI group and healthy control (HC) group were identified, with 7 immune cell types being significantly increased. Differences in the content of immune cells between different grades of SCI and the HC group were also discovered. DEIGs were identified, with alteration in some immune-related signaling pathways, vascular endothelial growth factor signaling pathways, and axon guidance signaling pathways. The SCI biomarkers identified and those of American Spinal Injury Society Impairment Scale (AIS) A and AIS D of SCI have certain diagnostic sensitivity. Analysis of the correlation of immune cells and biomarkers showed that biomarkers of SCI, AIS A grade and AIS D grade correlated positively or negatively with some immune cells. CKLF, EDNRB, FCER1G, SORT1, and TNFSF13B can be used as immune biomarkers for SCI. Additionally, GDF11and HSPA1L can be used as biomarkers of SCI AIS A grade; PRKCA and CMTM2 can be used as biomarkers of the SCI AIS D grade. Detecting expression of these putative biomarkers and changes in related immune cells may be helpful for predicting the severity of SCI.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.

Advancements in nanotechnology have promoted the rapid development of diverse nanosystems for biomedical applications. Manganese dioxide (MnO2) nanomaterials have attracted numerous attentions in the field of biosensing and cancer treatment due to their unique properties, such as simple fabrication procedures, broad optical absorption, and high oxidation and catalytic ability. Especially, the intelligent stimuli‐responsiveness of MnO2 nanomaterials to tumor microenvironment (TME) greatly improves their performance in tumor imaging and cancer therapy. In this review, the synthetic methods of different MnO2 nanostructures (nanoparticles, nanosheets, and nanoflowers) are first introduced, followed by the introduction of MnO2 nanomaterials for various biosensing applications based on their unique characteristics. Then, the recent achievements and progress of MnO2 nanomaterials for cancer treatment are comprehensively summarized with a focus on various therapeutic enhancements profiting from their disassembled ingredients under TME guidance. Finally, the current challenges and future perspectives of this promising research are discussed in the fields of biomedicine.

The therapeutic application of small interfering RNA (siRNA) is challenging due to its non-specific targeting and delivery issues. Here, the authors report an endogenous micro-RNA guided and hybridisation chain reaction-promoted siRNA delivery system encapsulated in tumour-derived extracellular vesicles, with cancer-specific activation, and achieve silencing of hypoxia-related genes.

Current DNA computational systems are constrained by integration efficiency, device structures and limited functions. Here the authors design a DNA arithmetic logic unit that uses polymerase-mediated strand displacement.

Berchemiella wilsonii (Rhamnaceae) is an endangered endemic tree to China. Here, the chloroplast genome was characterized by next-generation Illumina paired-end sequencing. The complete genome comprised 160,076 bp in length, with an inverted repeat region of 26,514 bp, a small single copy (SSC) region of 18,717 bp, and a large single copy region of 88,331 bp. A total of 132 genes were identified, including 83 protein-coding genes, 37 transfer RNA genes, and eight rRNA genes. The overall GC content of the whole genome was 37.23%. Phylogenetic analysis showed that B. wilsonii was most closely related to Berchemiella berchemiifolia.

The AC/DC hybrid microgrid has a large-scale and complex control process. It is of great significance and value to design a reasonable power coordination control strategy to maintain the power balance of the system. Based on hierarchical control, this paper designs a reasonable power coordination control strategy for AC/DC hybrid microgrid. For lower control, this paper designs a variety of control modes for each converter in different application scenarios. For the higher control, this paper analyzes the working mode of the system and designs the power coordination control strategy under the grid-connected and isolated island mode. In grid-connected operation, the DC bus voltage can be stabilized by adjusting the operation mode of the DC energy storage and the on-off of the secondary load. In isolated island operation, the DC sub-microgrid is the main microgrid, and the DC energy storage is the main power regulating equipment. This is based on the principle of “energy is in short supply in the system, DC energy storage finally discharge, energy supply exceeds demand in the system, DC energy storage gives priority to charging” of DC energy storage. By adjusting the control strategy of the micro-source, the reference power, and the on-off of the secondary load, the overall power balance is maintained. The Matlab/Simulink simulation software was used to build the AC/DC hybrid microgrid simulation model, which verified the effectiveness and stability of the proposed power coordination control strategy under various operating conditions.

The development of industry 4.0 has put forward higher requirements for modern milling technology. Monitoring the degree of milling tool wear during machine tool processing can improve product quality and reduce production losses. In the machining process of machine tools, many kinds of tools are usually used, and the signal characteristics of various sensors of different tools are different. Therefore, before the tool wear assessment, this paper identified the tool type according to the spindle current data. After the tool type recognition, this paper evaluates the tool wear degree according to the tool force data, vibration data, acoustic emission signal, and other multi-sensor data. Firstly, the Elman neural network and Adaboost algorithm are combined to construct the Elman_Adaboost strong predictor. Then, the variance and mean of seven sensor data were selected as the characteristic quantities to input the strong predictor. Finally, three wear quantities were obtained to measure the wear degree of the tool. The method proposed in this paper is implemented by Matlab, and the validity of this method is verified using the competition data provided by PHM (Prognostics and Health Management) Society. The results show that the average evaluation accuracy of the same tool wear is more than 92%, and that of the similar tool wear is more than 85%.

The sensing of pathogen infection and subsequent triggering of innate immunity are key to controlling zoonotic infections. Myxoma virus (MV) is a cytoplasmic DNA poxvirus that in nature infects only rabbits. Our previous studies have shown that MV infection of primary mouse cells is restricted by virus-induced type I interferon (IFN). However, little is known about the innate sensor(s) involved in activating signaling pathways leading to cellular defense responses in primary human immune cells. Here, we show that the complete restriction of MV infection in the primary human fibroblasts requires both tumor necrosis factor (TNF) and type I IFN. We also demonstrate that MV infection of primary human macrophages (pHMs) activates the cytoplasmic RNA sensor called retinoic acid inducible gene I (RIG-I), which coordinately induces the production of both TNF and type I IFN. Of note, RIG-I sensing of MV infection in pHMs initiates a sustained TNF induction through the sequential involvement of the downstream IFN-regulatory factors 3 and 7 (IRF3 and IRF7). Thus, RIG-I-mediated co-induction of TNF and type I IFN by virus-infected pHMs represents a novel innate defense mechanism to restrict viral infection in human cells. These results also reveal a new regulatory mechanism for TNF induction following viral infection.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assistedrecombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies. [ABSTRACT FROM AUTHOR]

There are five hot springs named separately SHANGT, ZHONGT, WENT, XIAT and SHENT in Lushan County, Pingdingshan City, Henan Province. The archaeal diversity of the five hot springs was investigated and analyzed. 16S rDNA v4-5 genes of arehaea were sequenced by lllumina Hisiq2500 PE250 technolog). The numbers of sample sequences and OTUs were sorted by using Uparse, Mothur, SILVA, MUSCLE and Qiime software. Finally, the archaeal abundance and diversity in the five hot springs were analyzed. 87 993, 67 326, 44 400, 48 611 and 48 495 effective reads have been obtained from SHANCT, ZHONCT, WENT, XIAT and SHENT respectively, and 520, 280, 678,451 and 437 OTUs have been acquired. The analysis of archaeal classification showed that the arehaea from the five hot springs could be classified into 11 phyla, in which Thaumarchaeota accounted for 95. 25% and was the dominant arehaea. In the genera level, the dominant arehaea of the SHANGT, ZHONGT and WENT was Candidalus-Nilrosocaldus, and unidentified-Thaumarchaeota was the dominant arehaea in XIAT and SHENT. The results showed that the five hot springs in Lushan are rich in microbial resources. The research provided basic data for further research and utilization of the arehaea resources in the five hot springs of Lushan. [ABSTRACT FROM AUTHOR]

The vibration signal of rolling bearing is usually complex and the useful fault information is hidden in the background noise, therefore, it is a challenge to identify rolling bearing faults from the complex vibration environment. In this paper, a novel multilayer deep learning convolutional neural network (CNN) method to identify rolling bearing fault is proposed. Firstly, in order to avoid the influence of different characteristics of the input data on the identification accuracy, a normalization preprocessing method is applied to preprocess the vibration signals of rolling bearings. Secondly, a multilayer CNN based on deep learning is designed in this paper to improve the fault identification accuracy of rolling bearing. Simulation data and experimental data analysis results show that the proposed method has better performance than SVM method and ANN method without any manual feature extractor design. [ABSTRACT FROM AUTHOR]

The progressive development of amplified DNA sensors using nucleic acid-based machineries, involving the isothermal autonomous synthesis of the Mg2+-dependent DNAzyme, is used for the amplified, multiplexed analysis of genes (Smallpox, TP53) and metal ions (Ag+, Hg2+). The DNA sensing machineries are based on the assembly of two sensing modules consisting of two nucleic add scaffolds that indude recognition sites for the two genes and replication tracks that yield the nicking domains for NtBbvCI and two different Mg2+-dependent DNAzyme sequences. In the presence of any of the genes or the genes together, their binding to the respective recognition sequences triggers the nicking/polymerization machineries, leading to the synthesis of two different Mg2+-dependent DNAzyme sequences. The cleavage of two different fluorophore/quencher-modified substrates by the respective DNAzymes leads to the fluorescence of F1 and/or F2 as readout signals for the detection of the genes. The detection limits for analyzing the Smallpox and TPS3 genes correspond to 0.1 nM Similarly, two different nucleic add scaffolds that indude Ag+-ions or Hg2+-ions recognition sequences and the replication tracks that yield the NtBbvCI nicking domains and the respective Mg2+-dependent DNAzyme sequences are implemented as niddng/replication machineries for the amplified, multiplexed analysis of the two ions, with detection limits corresponding to 1 nM. The ions sensing modules reveal selectivities dominated by the respective recognition sequences associated with the scaffolds. [ABSTRACT FROM AUTHOR]

The pH-controlled release of substrates from mesoporous SiO[sub 2] nanoparticles, MP-SiO[sub 2] NPs, is demonstrated by capping the pores with the Mg2+- or UO[sub 2]2+-dependent DNAzyme sequences and unlocking of the pores with Mg2+ ions or UO22+ ions at appropriate pH values. While the Mg2+-dependent DNAzyme reveals high activity at pH = 7.2, moderate activity at pH = 6.0, and it lacks activity at pH = 5.2, the UO22+-dependent DNAzyme reveals high activity at pH = 5.2, moderate activity at pH = 6.0, and it is catalytically inactive at pH = 7.2. Accordingly, the MP-SiO2 NPs were loaded with methylene blue, MB+, or thionine, Th+, and locked in the pores by the Mg2+- and UO22+-dependent DNAzyme sequences, respectively. The pH-programmed release of MB+ or Th+ from the loaded NPs proceeds, in the presence of Mg2+ ions or UO2+2 ions, at pH = 7.2 and pH = 5.2, using the Mg[sup 2+]- and UO2-dependent DNAzyme as catalysts that cleave the protecting caps and unlock the pores, respectively. At pH = 6.0 the MB+- and Th+-loaded NPs are concomitantly unlocked by the two DNAzymes. The unlocking processes are selective and other metal ions do not stimulate the release processes. [ABSTRACT FROM AUTHOR]

The pH-controlled release of substrates from mesoporous SiO2 nanoparticles, MP-SiO2 NPs, is demonstrated by capping the pores with the Mg2+- or UO22+-dependent DNAzyme sequences and unlocking of the pores with Mg2+ ions or UO22+ ions at appropriate pH values. While the Mg2+-dependent DNAzyme reveals high activity at pH = 7.2, moderate activity at pH = 6.0, and it lacks activity at pH = 5.2, the UO22+-dependent DNAzyme reveals high activity at pH = 5.2, moderate activity at pH = 6.0, and it is catalytically inactive at pH = 7.2. Accordingly, the MP-SiO2 NPs were loaded with methylene blue, MB+, or thionine, Th+, and locked in the pores by the Mg2+ - and UO22+-dependent DNAzyme sequences, respectively. The pH-programmed release of MB+ or Th[sup +] from the loaded NPs proceeds, in the presence of Mg2+ ions or UO22+ ions, at pH = 7.2 and pH = 5.2, using the Mg2+- and UO22+-dependent DNAzyme as catalysts that cleave the protecting caps and unlock the pores, respectively. At pH = 6.0 the MB+- and Th+-loaded NPs are concomitantly unlocked by the two DNAzymes. The unlocking processes are selective and other metal ions do not stimulate the release processes. [ABSTRACT FROM AUTHOR]

The article focuses on approaches in DNA nanostructures for activation of catalytic DNA cascades and use of the DNA nanostructures for control in reaction, sensing, logic operations, and assembly of complex nanostructures. Topics discussed include formation of DNA nanostructures by the autonomous hybridization chain reaction (HCR), use of catalytic nucleic acids DNAzymes in designing of logic gate systems and activation of catalytic cascade by coupling reaction of DNAzymes.

The amplified, highly sensitive detection of DNA using the dendritic rolling circle amplification (RCA) is introduced. The analytical platform includes a circular DNA and a structurally tailored hairpin structure. The circular nucleic acid template includes a recognition sequence for the analyte DNA (the Tay-Sachs mutant gene), a complementary sequence to the Mg2+-dependent DNAzyme, and a sequence identical to the loop region of the coadded hairpin structure. The functional hairpin in the system consists of the analyte-sequence that is caged in the stem region and a single-stranded loop domain that communicates with the RCA product. The analyte activates the RCA process, leading to DNA chains consisting of the Mg2+-dependent DNAzyme and sequences that are complementary to the loop of the functional hairpin structure. Opening of the coadded hairpin releases the caged analyte sequence, resulting in the dendritic RCA-induced synthesis of the Mg2+-dependent DNAzyme units. The DNAzyme-catalyzed cleavage of a fluorophore/quencher-modified substrate leads to a fluorescence readout signal. The method enabled the analysis of the target DNA with a detection limit corresponding to 1 aM. By the design of two different circular DNAs that include recognition sites for two different target genes, complementary sequences for two different Mg2+-dependent DNAzyme sequences and two different functional hairpin structures, the dendritic RCA-stimulated multiplexed analysis of two different genes is demonstrated. The amplified dendritic RCA detection of DNA is further implemented to yield the hemin/G-quadruplex horseradish peroxidase (HRP)-mimicking DNAzyme as catalytic labels that provide colorimetric or chemiluminescent readout signals. [ABSTRACT FROM AUTHOR]

The progressive development of amplified DNA sensors and aptasensors using replication/nicking enzymes/DNAzyme machineries is described. The sensing platforms are based on the tailoring of a DNA template on which the recognition of the target DNA or the formation of the aptamer-substrate complex trigger on the autonomous isothermal replication/nicking processes and the displacement of a Mg2+-dependent DNAzyme that catalyzes the generation of a fluorophore-labeled nucleic acid acting as readout signal for the analyses. Three different DNA sensing configurations are described, where in the ultimate configuration the target sequence is incorporated into a nucleic acid blocker structure associated with the sensing template. The target-triggered isothermal autonomous replication/nicking process on the modified template results in the formation of the Mg2+-dependent DNAzyme tethered to a free strand consisting of the target sequence. This activates additional template units for the nucleic acid self-replication process, resulting in the ultrasensitive detection of the target DNA (detection limit 1 aM). Similarly, amplified aptamer-based sensing platforms for cocaine are developed along these concepts. The modification of the cocaine-detection template by the addition of a nucleic acid sequence that enables the autonomous secondary coupled activation of a polymerization/nicking machinery and DNAzyme generation path leads to an improved analysis of cocaine (detection limit 10 nM). [ABSTRACT FROM AUTHOR]

Hybrid systems consisting of nucleic-acid-functionalized silver nanoclusters (AgNCs) and graphene oxide (GO) are used for the development of fluorescent DNA sensors and aptasensors, and for the multiplexed analysis of a series of genes of infectious pathogens. Two types of nucleic-acid-stabilized AgNCs are used: one type includes the red-emitting AgNCs (616 nm) and the second type is near-infrared-emitting AgNCs (775 nm). Whereas the nucleic-acid-stabilized AgNCs do not bind to GO, the conjugation of single-stranded nucleic acid to the DNA-stabilized AgNCs leads to the adsorption of the hybrid nanostructures to GO and to the fluorescence quenching of the AgNCs. By the conjugation of oligonucleotide sequences acting as probes for target genes, or as aptamer sequences, to the nucleic-acid-protected AgNCs, the desorption of the probe/nucleic-acid-stabilized AgNCs from GO through the formation of duplex DNA structures or aptamer–substrate complexes leads to the generation of fluorescence as a readout signal for the sensing events. The hybrid nanostructures are implemented for the analysis of hepatitis B virus gene (HBV), the immunodeficiency virus gene (HIV), and the syphilis (Treponema pallidum) gene. Multiplexed analysis of the genes is demonstrated. The nucleic-acid-AgNCs-modified GO is also applied to detect ATP or thrombin through the release of the respective AgNCs-labeled aptamer–substrate complexes from GO. [ABSTRACT FROM AUTHOR]

The Zn2+-dependent ligation DNAzyme is implemented as a biocatalyst for the amplified detection of a target DNA by the autonomous replication of a nucleic acid reporter unit that is generated by the catalyzed ligation process. The reporter units enhance the formation of active DNAzyme units, thus leading to the isothermal auto-catalytic formation of the reporter elements. The system was further developed and applied for the amplified detection of Tay-Sachs genetic disorder mutant, with a detection limit of 1.0 × 10-11 M. Besides providing a versatile paradigm for the amplified detection of DNA, the system reveals a new, enzyme-free, isothermal, autocatalytic mechanism that introduces means for effective programmed synthesis. [ABSTRACT FROM AUTHOR]

An enzyme-free amplified detection platform is described using the horseradish peroxidase (HRP)-mimicking DNAzyme as an amplifying label. Two hairpin structures that include three-fourths and one-fourth of the HRP-mimicking DNAzyme in caged, inactive configurations are used as functional elements for the amplified detection of the target DNA. In the presence of the analyte DNA, one of the hairpins is opened, and this triggers the autonomous cross-opening of the two hairpins using the strand displacement principle. This leads to the formation of nanowires consisting of the HRP-mimicking DNAzyme. The resulting DNA nanowires act as catalytic labels for the colorimetric or chemiluminescent readout of the sensing processes (the term "enzyme-free" refers to a protein-free catalyst). The analytical platform allows the sensing of the analyte DNA with a detection limit corresponding to 1 x 10-13 M. The optimized system acts as a versatile sensing platform, and by coaddition of a "helper" hairpin structure any DNA sequence may be analyzed by the system. This is exemplified with the detection of the BRCA1 oncogene with a detection limit of 1 x 10-13 M. [ABSTRACT FROM AUTHOR]

Biological systems that are capable of performing computational operations could be of use in bioengineering and nanomedicine, and DNA and other biomolecules have already been used as active components in biocomputational circuits. There have also been demonstrations of DNA/RNA-enzyme-based automatons, logic control of gene expression, and RNA systems for processing of intracellular information. However, for biocomputational circuits to be useful for applications it will be necessary to develop a library of computing elements, to demonstrate the modular coupling of these elements, and to demonstrate that this approach is scalable. Here, we report the construction of a DNA-based computational platform that uses a library of catalytic nucleic acids (DNAzymes), and their substrates, for the input-guided dynamic assembly of a universal set of logic gates and a half-adder/half-subtractor system. We demonstrate multilayered gate cascades, fan-out gates and parallel logic gate operations. In response to input markers, the system can regulate the controlled expression of anti-sense molecules, or aptamers, that act as inhibitors for enzymes. [ABSTRACT FROM AUTHOR]

In this contribution we illustrate a simple layer-by-layer assembly strategy to immobilize DNA on the surface of gold thin film and to trigger the selective release of DNA upon the exclusive control of external electric potential. The assembly of DNA-containing multilayer films is driven by coordination/electrostatic interactions between inorganic zirconium ion (Zr4+) and phosphate groups in the backbone of the DNA chain. The selective electrodissolution of the film is might attributable to the migration of ions in the multilayer film and the electrodeposition of ions on the gold surface, as demonstrated by X-Ray photoelectron spectroscopy (XPS) and atomic emission spectroscopy (AES). Further electrochemical surface plasmon resonance (EC-SPR) results demonstrate that the selective electrodissolution profiles of the film are depended on both of the applied potential and the ionic strength of electrolyte. The electrodissolution process can be switched off by removing the external electric potential and reactivated by reapplying the external electric potential under physiological pH condition. By incorporating plasmid DNA (pDNA) in to this system, the multilayer films can sustain the consecutive release of pDNA with its integrity and transcriptional activity retained. Given the non-specific nature of the electrostatic interactions the approach presented herein may, in principle, also be extended from polynucleotides to other negatively charged biomolecules, making this selective electrodissolution appealing model system for biomedical applications where spatiotemporal control of biological material is required. [ABSTRACT FROM AUTHOR]

A layer-by-layer film composed of DNA and inorganic zirconium ion (Zr 4+) was fabricated on the surface of gold thin film, and an electric field triggered disintegration of the multilayer film was studied by using electrochemical surface plasmon resonance (EC-SPR). EC-SPR results demonstrated that the film was disassembled upon the application of an electric field and the disassembly rate varied with the applied potential, leading to the controlled release of DNA. The electrodissolution could be switched off by removing the electric potential and reactivated by reapplying the potential. By incorporating plasmid DNA (pDNA) in to this controlled release system, the multilayer film could sustain the consecutive release of pDNA electrochemically. The released pDNA retained its integrity and transfection activity, and expressed enhanced green fluorescent protein (EGFP) after being transfected into HEK 293 cells. The electrochemical systems, with advantages of miniaturization, surface-tailoring, safety, simplicity, convenience, automation, low-cost, and free of immune reactions, made the electrical route a very attractive gene-delivery alternative. [ABSTRACT FROM AUTHOR]

The authors have recently demonstrated that wild-type myxoma virus (MV) tagged with gfp (vMyxgfp) can generate a tumor-specific infection that productively infects and clears human tumor-derived xenografts when injected intratumorly into human gliomas transplanted into immunodeficient mice (Lun et al, 2005). To expand the understanding of MV tropism in cancer cells from a specific tissue lineage, the authors have screened a series of human glioma cells (U87, U118, U251, U343, U373) for myxoma virus replication and oncolysis. To assess the viral tropism determinants for these infections, the authors have screened myxoma virus knockout constructs that have been deleted for specific host range genes (M-T2, M-T4, M-T5, M11L, and M063), as well as a control MV gene knockout construct with no known host range function (vMyx135KO) but is highly attenuated in rabbits. The authors report wide variation in the ability of various vMyx-hrKOs to replicate and spread in the human glioma cells as measured by early and late viral gene expression. This differential ability to support vMyx-hrKO productive viral replication is consistent with levels of endogenous activated Akt in the various gliomas. The authors have identified one vMyx-hrKO virus (vMyx63KO) and one nonhost range knockout construct (vMyx135KO) that appear to replicate in the gliomas even more efficiently than the wild-type virus and that reduce the viability of the infected gliomas. These knockout viruses also inhibit the proliferation of gliomas in a manner similar to the wild-type virus. Together these data, as well as the fact that these knockout viruses are attenuated in their natural hosts, may represent environmentally safer candidate oncolytic viruses for usage in human trials. [ABSTRACT FROM AUTHOR]

A systematic study of the amplified optical detection of DNA by Mg2+-dependent DNAzyme subunits is described. The use of two DNAzyme subunits and the respective fluorophore/quencher-modified substrate allows the detection of the target DNA with a sensitivity corresponding to 1 × 10-9 M. The use of two functional hairpin structures that include the DNAzyme subunits in a caged, inactive configuration leads, in the presence of the target DNA, to the opening of one of the hairpins and to the activation of an autonomous cross-opening process of the two hairpins, which affords polymer DNA wires consisting of the Mg2+-dependent DNAzyme subunits. This amplification paradigm leads to the analysis of the target DNA with a sensitivity corresponding to 1 × 10-14 M. The amplification mixture composed of the two hairpins can be implemented as a versatile sensing platform for analyzing any gene in the presence of the appropriate hairpin probe. This is exemplified with the detection of the BRCA1 oncogene. [ABSTRACT FROM AUTHOR]