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The capacity to retain and precisely release historical data at the right moments is typically managed by sequential logic circuits within computer systems. However, the reusability and autonomy of DNA sequential logic circuits still need to be developed. To bridge this gap, a series of sequential logic circuits are implemented by constructing a DNA strand replacement system regulated by a nicking enzyme (nickase). This nickase‐integrated system dynamically resolves the thermodynamic‐kinetic conflict, offering spatiotemporal control over strand displacement. These circuits include Set‐Reset latches (SR‐latches) constructed with NOR and NAND gates, along with Data latches (D‐latches), are designed with simplicity, autonomy, and reusability. Furthermore, addition, subtraction, and reversible counters leveraging these foundational circuits are successfully constructed. These latches are further applied to transient miRNA recording, environmental toxin detection, and real‐time ATP imaging in living cells. In this work, a DNA sequential logic circuit is constructed that combines reusability and autonomy for the first time, including SR (Set‐Reset)‐latches composed of NOR gates, SR‐latches composed of NAND gates, and D (Data)‐latches. After fully utilizing the characteristics of each latch, to counter, toxin and miRNA detection and intracellular dynamic ATP monitoring are successfully applied.

Alkaline phosphatase (ALP) is widely expressed in human tissues. ALP plays an important role in the dephosphorylation of proteins and nucleic acids. Therefore, quantitative analysis of ALP plays a vital role in disease diagnosis and the development of biological detection methods. Terminal deoxynucleotidyl transferase (TdT) catalyzes continuous polymerization of deoxynucleotide triphosphates at the 3′-OH end of single-stranded DNA in the absence of a template. In this study, we developed a highly sensitive and selective method based on TdT and endonuclease IV (Endo IV) to quantify ALP activity. After ALP hydrolyzes the 3′-PO4 end of the substrate and generates 3′-OH, TdT can effectively elongate the 3′-OH end with deoxynucleotide adenine triphosphate (dATP) and produce a poly A tail, which can be detected by the poly T probes. Endo IV digests the AP site in poly T probes to generate a fluorescent signal and a new 3′-OH end, leading to the generation of exponential fluorescence signal amplification. The substrate for TdT elongation was optimized, and a limit of detection of 4.3 × 10−3 U/L was achieved for ALP by the optimized substrate structure. This method can also detect ALP in the cell lysate of a single cell. This work has potential applications in disease diagnosis and biomedical detection. [Display omitted] •A highly sensitive fluorescence assay was developed for detecting ALP activity.•ALP triggered the exponential signal amplification with TdT and Endo IV.•ALP activity was detected in cell lysates containing as few as one cell.•Substrate structure preference of TdT was investigated to enhance assay sensitivity.

Abstract Background Sperm DNA integrity is crucial for normal fertilization, implantation, and embryo development. Several assays are available to assess sperm DNA fragmentation but are limited by high price, complicated processes, and low accuracy. Methods We developed a secondary amplification detection system based on terminal deoxynucleotidyl transferase and endonuclease IV, which could efficiently measure the number of 3'-OH (equivalent to the number of breakpoints). We applied this detection system in single stranded DNA with standard concentrations to obtain the standard curve. We then broke the double stranded genomic DNA by ultrasound and enzyme digestion and used the detection system to monitor the increase of DNA breakpoints. Finally, we used this method to measure the mean number of sperm DNA breakpoints (MDB) in 80 sperm samples. Results We successfully measured the number of 3'-OH in single stranded DNA with standard concentration and obtained the standard curve. The linear range for the number of DNA breakpoints was from 0.1 nM to 15 nM. The detection method was successfully validated on λ DNA and 80 human sperm samples. The results of real clinical samples revealed that the mean number of DNA breakpoints (MDB) had a stronger relevance with the sperm motility and clinical pregnancy outcomes than the commonly used parameter of DNA fragmentation index (DFI). Conclusion We have developed a straight-forward method for direct measurement of the mean number of DNA breakpoints in sperms. The method has advantages of short time-consumption, simple operation, high analytical sensitivity, and low requirement for instrumentation, which makes it conducive to clinical application. The proposed new parameter (MDB) could be a more direct, accurate and clinically significant indicator for evaluating the sperm DNA integrity.

The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods. DNA mutations occurring at low frequencies can be more easily detected by first excising wild-type DNA strands via the endonuclease deoxyribonuclease I, guided by single-stranded phosphorothioated DNA.

Indole-3-acetyl- myo -inositol (IAInos) is one of the most important auxin conjugates for storage and transportation of auxin. The information of its composition, distribution, and metabolism is particularly desired for elucidating the related signal transduction pathways of the plant hormones. However, separation and quantification of the four individual IAInos isomers in plant tissues have not been reported so far. In this work, we first synthesized and isolated four IAInos isomers using semi-preparative high-performance liquid chromatography (HPLC). The IAInos isomer structures were characterized using liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC-QTOF/MS) and nuclear magnetic resonance spectroscopy (NMR). Using these pure compounds as internal or external standards, an efficient LC-MS method was developed for simultaneous detection of indole-3-acetic acid, methyl indole-3-acetic acid ester, and the four IAInos isomers in plant tissue samples. The linear working range and lower limit of detection for the four IAInos isomers are 10–2,000 ng mL −1 and 5.0 ng mL −1 , respectively. The stabilities and interconversion pathways of IAInos isomers were studied using our synthetic isomers. It was found that two IAInos isomers existed in Zea mays kernels, while all of the four IAInos isomers were present in the roots of Arabidopsis thaliana . The content of IAInos in A. thaliana roots was much lower than in the Z. mays kernels. The methodology in this article provides useful techniques and methods for systematic study on the phytophysiology and phytochemistry of IAA conjugates and other related plant hormones. Figure Separation of the four isomers of indole-3-acetyl- myo -inositol

In this review, we summarize recent advances in the development of molecular recognition components based on the biomolecular scaffolds of proteins and nucleic acids for specific recognition of miscellaneous targets. In addition to the widely adopted recombinant antibody fragments, designed ankyrin repeat proteins and modular peptide repeats of transcription-activator-like effectors for base-specific recognition of DNA sequence are also briefly introduced. For the nucleic acid based molecular recognition systems, aptamers, including slow off-rate modified aptamers, DNAzymes, and synthetic DNA-like oligomers for versatile biorecognition are described. Finally, we discuss the remaining challenges and future research directions in the field.

Indole-3-acetyl-myo-inositol (IAInos) is one of the most important auxin conjugates for storage and transportation of auxin. The information of its composition, distribution, and metabolism is particularly desired for elucidating the related signal transduction pathways of the plant hormones. However, separation and quantification of the four individual IAInos isomers in plant tissues have not been reported so far. In this work, we first synthesized and isolated four IAInos isomers using semi-preparative high-performance liquid chromatography (HPLC). The IAInos isomer structures were characterized using liquid chromatography-electrospray ionization quadrupole time-of-flight tandem mass spectrometry (LC-QTOF/MS) and nuclear magnetic resonance spectroscopy (NMR). Using these pure compounds as internal or external standards, an efficient LC-MS method was developed for simultaneous detection of indole-3-acetic acid, methyl indole-3-acetic acid ester, and the four IAInos isomers in plant tissue samples. The linear working range and lower limit of detection for the four IAInos isomers are 10-2,000 ng m[L.sup.-1] and 5.0 ng m[L.sup.-1], respectively. The stabilities and interconversion pathways of IAInos isomers were studied using our synthetic isomers. It was found that two IAInos isomers existed in Zea mays kernels, while all of the four IAInos isomers were present in the roots of Arabidopsis thaliana. The content of IAInos in A. thaliana roots was much lower than in the Z. mays kernels.

In this review, we summarize recent advances in the development of molecular recognition components based on the biomolecular scaffolds of proteins and nucleic acids for specific recognition of miscellaneous targets. In addition to the widely adopted recombinant antibody fragments, designed ankyrin repeat proteins and modular peptide repeats of transcription-activator-like effectors for base-specific recognition of DNA sequence are also briefly introduced. For the nucleic acid based molecular recognition systems, aptamers, including slow off-rate modified aptamers, DNAzymes, and synthetic DNA-like oligomers for versatile biorecognition are described. Finally, we discuss the remaining challenges and future research directions in the field.

The performance of electrodes is the most critical factor determining the output characteristics of high-temperature proton exchange membrane fuel cells (HT-PEMFCs), and the electrode structure directly determines the strength of mass transfer and electrochemical reactions. Therefore, exploring the mechanism of increasing the specific surface area of electrodes is crucial for the design of electrode structures. In this paper, the electrochemical characteristics and mass transport of an HT-PEMFC are investigated based on a three-dimensional single-channel model, and a mathematical model of the fin structure on the electrode surface is established to make comparisons with calculations. The results indicate that the oxygen mole concentration decreases with an increase in fin density. Meanwhile, the fuel cell reaches optimal performance at a low operating voltage and in high fin density conditions. In addition, the output performance of the PEMFC increases with the aspect ratio. Finally, the potential distribution of the simulation results coincides with the theoretical model, and the mechanism of electrode polarization on the performance of fin geometry can significantly support the interpretation of kinetic characteristics obtained from simulations. The research result contributes to the efficient design and preparation of future electrode structures of HT-PEMFCs.

Oily sludge, as a critical hazardous waste, requires appropriate treatment for resource recovery and harmfulness reduction. Here, fast microwave-assisted pyrolysis (MAP) of oily sludge was conducted for oil removal and fuel production. The results indicated the priority of the fast MAP compared with the MAP under premixing mode, with the oil content in solid residues after pyrolysis reaching below 0.2%. The effects of pyrolysis temperature and time on product distribution and compositions were examined. In addition, pyrolysis kinetics can be well described using the Kissinger-Akahira-Sunose (KAS) and the Flynn-Wall-Ozawa (FWO) methods, with the activation energy being 169.7–319.1 kJ/mol in the feedstock conversional fraction range of 0.2–0.7. Subsequently, the pyrolysis residues were further treated by thermal plasma vitrification to immobilize the existing heavy metals. The amorphous phase and the glassy matrix were formed in the molten slags, resulting in bonding and, hence, immobilization of heavy metals. Operating parameters, including working current and melting time, were optimized to reduce the leaching concentrations of heavy metals, as well as to decrease their volatilization during vitrification.