Spatiotemporal Visualization of Paraquat Distribution, Toxicokinetics, and Its Detoxification in Zebrafish Using Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging.

Paraquat is a highly toxic quaternary ammonium herbicide. It can damage the functions of multiple organs and cause irreversible pulmonary fibrosis in the human body. However, the toxicological mechanism of paraquat is not yet fully understood, and due to the lack of specific antidotes, the clinical treatment of paraquat intoxication is still a great medical challenge. In-depth research on its toxicity mechanism, toxicokinetics, and effective antidotes is urgently demanded. A new molecular imaging technique, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), can simultaneously achieve quantitative and spatial analysis and offer an alternative, distinct, and useful technique for paraquat intoxication and consequent detoxication. Here, we visualized the spatial-temporal distribution and conducted toxicokinetic research on paraquat in zebrafish by using stable isotope-labeled internal-standard-aided MALDI-MSI for the first time. The results indicated that paraquat had a fast absorption rate and was widely distributed in different organs, such as the brain, gills, kidneys, and liver in zebrafish. Its half-life was long, and the elimination rate was slow. Paraquat reached its peak at 30 min and was mainly distributed in kidneys and intestines and then showed a tendency of declining first but mildly rising later at 6 h, accompanied by a wide distribution in kidneys and intestines again. It suggested that entero-systemic recirculation might lead to the observed secondary peaks, and perhaps it extended the residence time of paraquat in the body. In addition, we validated the potential detoxification effect of sodium salicylate as a potential antidote for paraquat from both the dimensions of distribution and quantification. In conclusion, MALDI-MSI conveniently provided the distinct and quantitative spatial-temporal distribution information on paraquat in the whole body of zebrafish; it will promote the understanding of its toxicokinetic characteristics and provide more valuable information for clinical treatment.

Impaired Efferocytosis Enables Apoptotic Osteoblasts to Escape Osteoimmune Surveillance During Aging.

Macrophage efferocytosis of apoptotic osteoblasts (apoOBs) is a key osteoimmune process for bone homeostasis. However, apoOBs frequently accumulate in aged bone marrow, where they may mount proinflammatory responses and progressive bone loss. The reason why apoOBs are not cleared during aging remains unclear. In this study, it is demonstrated that aged apoOBs upregulate the immune checkpoint molecule CD47, which is controlled by SIRT6-regulated transcriptional pausing, to evade clearance by macrophages. Using osteoblast- and myeloid-specific gene knockout mice, SIRT6 is further revealed to be a critical modulator for apoOBs clearance via targeting CD47-SIRPα checkpoint. Moreover, apoOBs activate SIRT6-mediated chemotaxis to recruit macrophages by releasing apoptotic vesicles. Two targeting delivery strategies are developed to enhance SIRT6 activity, resulting in rejuvenated apoOBs clearance and delayed age-related bone loss. Collectively, the findings reveal a previously unknown linkage between immune surveillance and bone homeostasis and targeting the SIRT6-regulated mechanism can be a promising therapeutic strategy for age-related bone diseases.

A spatially-controlled DNA triangular prism nanomachine for AND-gated intracellular imaging of ATP in acidic microenvironment.

A DNA triangular prism nanomachine (TPN)-based logic device for intracellular AND-gated imaging of adenosine triphosphate (ATP) has been constructed. By using i-motif sequences and ATP-binding aptamers as logic control units, the TPN logic device is qualified to respond to the acidic environment and ATP in cancer cell lysosomes. Once internalized into the lysosome, the specific acidic microenvironment in lysosome causes the i-motif sequence to fold into a tetramer, resulting in compression of DNA tri-prism. Subsequently, the split ATP aptamer located at the tip of the collapsed triangular prism binds stably to ATP, which results in the fluorescent dyes (Cy3 and Cy5) modified at the ends of the split aptamer being in close proximity to each other, allowing Förster Resonance Energy Transfer (FRET) to occur. The FRET signals are excited at a wavelength of 543 nm and can be collected within the emission range of 646-730 nm. This enables the precise imaging of ATP within a cell. We also dynamically operate AND logic gates in living cells by modulating intracellular pH and ATP levels with the help of external drugs. Owing to the AND logic unit on TPN it can simultaneously recognize two targets and give corresponding intelligent logic judgment via imaging signal output. The accuracy of molecular diagnosis of cancer can be improved thus eliminating the false positive signal of single target-based detection. Hence, this space-controlled TPN-based logical sensing platform greatly avoids sensitivity to extracellular targets during the cell entry process, providing a useful tool for high-precision imaging of the cancer cell's endogenous target ATP.

Combination of specific proteins as markers for accurate detection of extracellular vesicles using proximity ligation-mediated bHCR amplification.

As the molecular characteristics of extracellular vesicles (EVs) are closely related to the occurrence and progression of cancer, the detection of tumor-derived EVs provides a promising non-invasive tool for the early diagnosis and treatment of cancer. However, it would be difficult for most of the existing methods to avoid false positives because the obtained result declares the amounts of proteins, but cannot accurately reflect the protein sources, including EV proteins and interfering proteins, in the actual samples. In this manuscript, a robust, accurate, and sensitive fluorescent strategy for profiling EV proteins is developed by using the combination of specific proteins as markers (Co-marker). Our strategy relies on the Co-marker recognition-activated cascade bHCR amplification, which forms numerous G-quadruplex structures that are integrated with fluorescent dyes for signal transduction. Notably, the detection accuracy can be improved owing to the effective avoidance of false positives from interfering proteins or single protein markers. Moreover, by using the double-positive protein recognition mode, unpurified detection can be achieved that avoids time-consuming EVs purification procedures. With its capacities of accuracy, portability, sensitivity, high throughput, and non-purification, the developed strategy might provide a practical tool for EV identification and the related early diagnosis and treatment of cancer.

DNA tetrahedron-besieged primer and DNAzyme-activated programmatic RCA for low-background electrochemical detection of ochratoxin A.

Ochratoxin A (OTA) is the most toxic class of ochratoxins and has become a major threat to the environment, humans and animals. Therefore, research on the methods for its detection is also more urgent. Herein, we propose a low-background electrochemical biosensor based on a DNA tetrahedron-besieged primer and a DNAzyme-activated programmatic rolling circle amplification (RCA) that can be ultimately utilized for OTA detection in wine samples. Low-background detection can be achieved using the besieged primer via sequenced assembly of DNA tetrahedral nanostructures so that non-specific extensions of primer can be avoided. The target OTA-mediated DNAzyme activation initiates the programmatic RCA. Additionally, the catalytic property of silver nanoclusters (AgNCs) is integrated with the electrochemical assay to achieve high sensitivity for OTA detection. Benefiting from the aforementioned processes, a low-background, and highly sensitive electrochemical biosensor has been successfully constructed. This design is capable of detecting OTA at concentrations from 1 pg/mL to 10 ng/mL, and its lowest concentration limit is 0.773 pg/mL. Simultaneously, its validation in the detection of actual samples reveals that the proposed electrochemical biosensor has a lot of potential in food safety and environmental detection.

An autonomous synthetic DNA machine for ultrasensitive detection of Salmonella typhimurium based on bidirectional primers exchange reaction cascades.

Statistics show that food poisoning caused by Salmonella typhimurium (S. Typhimurium) often tops the list of bacterial food poisoning types in countries around the world. However, detecting traces of S. Typhimurium in real samples remains challenging. In recent years, primer exchange reaction (PER), a new isothermal amplification strategy, has rapidly attracted the attention of researchers in the field of biosensing. In this work, We developed a nanostructure called DNA arch bridge (DAB) and combined the DAB with cascade PER technology to construct a novel bidirectional PER (B-PER) for ultra-sensitive detection of pathogenic bacteria as a novel fluorescent biosensor. This strategy relies on the B-PER reaction mediated by binding of the target and adaptor, which occurs with the assistance of Klenow Fragment (KF) (3'-5'exo) polymerase and produces a good deal of G-quadruplex sequences that generate a fluorescent signal by embedding fluorescent dyes. Under the best conditions, the biosensor achieves ultrasensitive detection of S. Typhimurium, and the detection limit of the strategy is 9.3 cfu mL-1 over the linear detection scope of 101-105 cfu mL-1. The method has the merits of facile operation, rapid response, and high sensitivity. Furthermore, the biosensor is expected to achieve ultrasensitive detection of various small molecules through recognizing different target and primer sequences. Therefore, our proposed strategy provides an efficient, stable, universal, and practical sensing platform for pathogen and other small molecules detection.

Non-destructive detection and classification of textile fibres based on hyperspectral imaging and 1D-CNN.

Textile fibre is very common in daily life, and its classification and identification play an important role in textile recycling, archaeology, public security, and other industries. However, traditional identification methods are time-consuming, laborious, and often destructive to the samples. In order to quickly, accurately, and nondestructively classify and recognize textile fibres, this study established a textile fibre classification and recognition method based on hyperspectral imaging (HSI) and a one-dimensional convolutional neural network (1D-CNN) model. Hyperspectral images of 25 kinds of commercial textile fibres were collected and denoised by pixel fusion. Four traditional machine learning classification models, k-nearest neighbors (KNN), support vector machine (SVM), random forest (RF), and partial least squares-discriminant analysis (PLS-DA), were used to identify the data. The results show that RF has the highest classification accuracy, reaching 91.4%. Then a back propagation neural network (BPNN) model and a one-dimensional convolutional neural network (1D-CNN) model were constructed and compared with the traditional machine learning methods. The results show that the 1D-CNN models have 97.9% and 98.6% accuracy on the training and test sets, respectively. The precision (Pr), sensitivity (Se), specificity (Sp), and F1 score (F1 score) of the models reached 98.7%, 98.6%, 99.9%, and 98.6%, respectively, which were significantly better than the four traditional machine learning models. It seems that 1D-CNN combined with the HSI technique may be a potential method in the detection and classification of textile fibres.

Target-manipulated drawstring DNAzyme for ultrasensitive detection of UDG using Au@Ag NRs indicator.

Uracil-DNA glycosylase (UDG) is a common glycosylase that can expressly recognize and remove damaged uracil bases, and the ultrasensitive detection of which is significant to maintain genomic stability and early clinical diagnosis of disease. Herein, we proposed a sensitive colorimetric sensing platform to detect UDG. Combined with target-manipulated drawstring DNAzyme and Au@Ag nanorods (Au@Ag NRs) indicator, we achieved in naked-eyes observation and ultrasensitive detection of UDG. Briefly, when the UDG exists, the dynamic reaction of rope pulling will occur generating the active conformation of DNAzyme. The cutting effect will be further produced when we add Mg2+, thus the generated trigger chain can mediate the occurrence of CHA reaction, followed by generating amount of ·OH which can etch Au@Ag NRs causing the shifted of localized surface plasmon resonance (LSPR) peak. By contrast, there is no obvious shift of LSPR peak. This strategy shows extraordinary specificity and sensitivity toward UDG providing a detection limit of 4.6 × 10-5 U mL-1. By using of this method, we detected UDG specifically in complex samples, proving that it's potential applications in biomedical research and clinical diagnosis are fantastic.

DNAzyme-controlled plasmonic coupling for SERS-based determination of Salmonella typhimurium using hybridization chain reaction self-assembled G-quadruplex.

A facile and rapid SERS strategy for S. typhimurium detection based on hybridization chain reaction (HCR) self-assembled G-quadruplex DNAzyme (GQH DNAzyme)-controlled plasmonic coupling was developed. GQH DNAzyme is introduced as a biocatalyst to catalyze the oxidation of L-cysteines to cysteines (thiols to disulfides) to assist SERS signal transduction. This is the first time that the self-assembled split GQH DNAzyme-controlled plasmonic coupling is integrated with SERS sensing. The results reveal the proposed SERS strategy can quantify S. typhimurium with a wide linear range (5 to 105 cfu mL-1) and a low detection limit (4 cfu mL-1; n = 5, mean ± standard deviation) and RSD of 7%. The method exhibited preeminent detection performance in spiked samples with recoveries of 93.1-117%. The proposed strategy has great potential for being a versatile SERS platform for detecting a wide spectrum of analytes by replacing them with the corresponding recognition elements. Therefore, this study not only creates a practical platform for pathogenic bacteria identification and related food safety testing and environmental monitoring, but also provides a new paradigm for building SERS sensor. A facile and rapid SERS strategy for S. Typhimurium detection based on hybridization chain reaction (HCR) self-assembled G-quadruplex DNAzyme-controlled plasmonic coupling.

Two-stage nicking enzyme signal amplification (NESA)-based biosensing platform for the ultrasensitive electrochemical detection of pathogenic bacteria.

The sensitive and selective detection of pathogenic bacteria represents an essential approach in food safety analysis and clinical diagnostics. We report the development of a simple, rapid, and low-cost electrochemical biosensing strategy for the detection of pathogenic bacteria with ultrasensitivity and high specificity. The biosensor relies on the target and aptamer binding-triggered two-stage nicking enzyme signal amplification (NESA) and three-way junction probe-mediated electrochemical signal transduction. In the presence of the target S. typhimurium, the specific binding of S. typhimurium and aptamer results in the release of a primer, which hybridizes with HAP1 and initiates an extension reaction with the aid of polymerase and dNTPs. A specific recognition site for Nt.BsmaI is generated in the DNA duplex; thus, the produced DNA is nicked and the secondary primer is released (named recycle I). Subsequently, the reaction solution supplemented with a helper DNA is dropped on the electrode surface, and a three-way junction probe containing a specific recognition site for Nt.BsmaI is thus formed. The MB-labeled probe is nicked with the help of Nt.BsmaI and the dissociated primer-helper DNA duplex combines with another HAP2 (named recycle II). Thus, a remarkably decreased electrochemical signal is generated because the electroactive MB is far away from the electrode surface. As far as we know, this work is the first time that NESA and three-way junction probe-mediated electrochemical signal transduction has been used for pathogenic bacteria detection. Under optimal conditions, the results reveal that the calibration plot obtained for S. typhimurium is approximately linear from 9.6 to 9.6 × 105 cfu mL-1 with the limit of detection of 8 cfu mL-1. Additionally, the proposed strategy has been successfully applied to the quantitative assay of S. typhimurium in the real samples. Therefore, the NESA-based biosensing strategy might create a useful and practical platform for pathogenic bacteria identification, and the related food safety analysis and clinical diagnosis.

Ultrasensitive Uracil-DNA Glycosylase Activity Assay and Its Inhibitor Screening Based on Primer Remodeling Jointly via Repair Enzyme and Polymerase.

The development of isothermal nucleic acid amplification techniques has great significance for highly sensitive biosensing in modern biology and biomedicine. A facile and robust exponential rolling circle amplification (RCA) strategy is proposed based on primer-remodeling amplification jointly via a repair enzyme and polymerase, and uracil-DNA glycosylase (UDG) is selected as a model analyte. Two kinds of complexes, complex I and complex II, are preprepared by hybridizing a circular template (CT) with a uracil-containing hairpin probe and tetrahydrofuran abasic site mimic (AP site)-embedded fluorescence-quenched probe (AFP), respectively. The target UDG specifically binds to complex I, resulting in the generation of an AP site, followed by cleavage via endonuclease IV (Endo IV) and the successive trimming of unmatched 3' terminus via phi29 DNA polymerase, thus producing a useable primer-CT complex that actuates the primary RCA. Then, numerous complex II anneal with the first-generation RCA product (RP), generating a complex II-RP assembly containing AP sites within the DNA duplex. With the aid of Endo IV and phi29, AFP, as a pre-primer in complex II, is converted into a mature primer to initiate additional rounds of RCA. So, countless AFPs are cleaved, releasing remarkably strong fluorescent signals. The biosensor is demonstrated to enable rapid and accurate detection of the UDG activity with an improved detection limit as low as 4.7 × 10-5 U·mL-1. Moreover, this biosensor is successfully applied for UDG inhibitor screening and complicated biological samples analysis. Compared to the previous exponential RCA methods, our proposed strategy offers additional advantages, including excellent stability, optional design of CT, and simplified operating steps. Therefore, this proposed strategy may create a useful and practical platform for ultrasensitive detection of low levels of analytes in clinical diagnosis and fundamental biomedicine research.

Effect of ammonium acetate on alcohol fermentation in cassava-alcohol fermentation process.

The cassava-alcohol fermentation process employing cassava requires nitrogen source to maximize yields by a commercial strain of S. cerevisiae TG1348. In this study, a factorial experimental design was used to assess a suitable nitrogen source for growth and fermentative performance of S. cerevisiae in cassava-ethanol fermentation. The alcohol fermentation time was about 39 h for urea and ammonium acetate, which was 48 h for ammonium chloride and ammonium sulphate. The fermentation time was reduced by 19 % when using urea and ammonium acetate as nitrogen source. Ammonium acetate leaded to the highest alcohol yield, which was 4% higher than for ammonium sulphate. In addition, byproduct formation differed obviously between the nitrogen sources. The glycerol yields were similar for urea, ammonium sulphate and ammonium chloride but were 24 % lower for ammonium acetate. However, glycerol yield for ammonium carbonate was higher than for other nitrogen sources. Clearly, in batch cultures the ammonium acetate not only increased ethanol generation, but also decreased glycerol generation. In order to understand why ammonium acetate promotes alcohol fermentation, acetic acid was added to different nitrogen sources. The weight loss effect of ammonium sulphate adding acetic acid and ammonium acetate as nitrogen source was the same. The fermentation time was shortened by adding acetic acid. And pH was increased by addition of acetic acid when ammonium sulfate and urea were used as nitrogen sources. The results showed that the acetate root plays an important role in ammonium acetate. The results of this study could facilitate the development of new strategies to control fermentation performance.

A three-dimensional dynamic DNA walker-mediated branching hybridization chain reaction for the ultrasensitive fluorescence sensing of ampicillin.

In this study, a novel, rapid and ultrasensitive fluorescence strategy using the three-dimensional (3D) dynamic DNA walker (DW)-induced branched hybridization chain reaction (bHCR) has been proposed for the detection of ampicillin (AMP). The sensing system was composed of an Nt·Bbvcl-powered DNA walker blocked by an AMP aptamer, hairpin-shaped DNA track probe (TP) and four kinds of metastable hairpin probes as the substrates of bHCR, which triggered the formation of the split G-quadruplex as the signal molecule. Due to the reasonable design, the specific binding between AMP and its aptamer activated the DW, and the DW moved on the surface of the gold nanoparticles (AuNPs) with the help of Nt·Bbvcl to produce primer probes (PPs), which induced bHCR. The products of the bHCR gathered two split G-quadruplex sequences together to form one complete G-quadruplex. The formed G-quadruplex emitted a strong fluorescence signal in the presence of thioflavin-T (ThT) to achieve the purpose of detecting AMP. The sensitivity of this method was greatly improved by the use of the 3D DNA walker and bHCR. The split G-quadruplex enhanced the signal-to-noise ratio (SNR). Under the optimal experimental conditions, a good correlation was obtained between the fluorescence intensity of the sensing system and the concentration of AMP ranging from 5 pM to 500 nM with a limit of detection (LOD) of 3.68 pM. Simultaneously, the method has been applied to the detection of antibiotics in spiked milk samples with satisfactory results.

Accurate and Nonpurified Identification of Extracellular Vesicles Using Dual-Binding Recognition Mode.

Circulating extracellular vesicles (EVs) are promising biomarkers for the early diagnosis and prognosis of cancer in a non-invasive manner. However, the rapid and accurate identification of EVs in complex biological samples is technically challenging, which is attributed to the requirement of extensive sample purification and unsatisfactory detection accuracy due to the disturbance of interfering proteins. Herein, a simultaneous binding of double-positive EV membrane protein-based recognition mode (DRM) is proposed. By the combination of DRM-mediated toehold activation and G-quadruplex DNAZyme-catalyzed etching of Au@Ag nanorods (Au@Ag NRs), we have developed an accurate, non-purified, low-cost, and visual strategy for EV identification. The synchronous binding of double-positive proteins on EV membranes is validated by confocal laser scanning microscopy analysis. This approach exhibits excellent specificity and sensitivity toward EVs ranging from 1.0 × 105 to 1.0 × 109 particles/mL with a detection limit of 6.31 × 104 particles/mL. Moreover, we have successfully realized non-purified EV quantification in complex biological media. In addition, target-initiated catalyzed hairpin assembly (CHA) is integrated with G-quadruplex DNAZyme-catalyzed color variation of Au@Ag NRs; thus, low-background EV detection can be achieved by the naked eye. Furthermore, our strategy is easy to adapt to high-throughput formats by using an automatic microplate reader, which could be expected to meet the requirements for high-throughput detection of clinical samples. With its capacities of rapidness, portability, affordability, high throughput, non-purification, and visual detection, this strategy could provide a practical tool for accurate identification of EVs and early diagnosis of cancer.

Target-swiped DNA lock for electrochemical sensing of miRNAs based on DNAzyme-assisted primer-generation amplification.

As an extremely important post-transcriptional regulator, miRNAs are involved in a variety of crucial biological processes, and the abnormal expressions of miRNAs are closely related to a variety of diseases. In this work, for the first time, we designed a nucleic acid lock nanostructure for specific detection of miRNA-21, which changes the self-structure to "active conformation" by binding the target, in order to generate triggers to initiate the subsequent reaction. Emphatically, this flexible nucleic acid lock is capable of self-cleaving without the assistance of external component, overcoming the disadvantages of the complex design and requiring protease assistance in traditional nanostructure. Moreover, the combination of DNAzyme and RCA technology not only greatly improves the efficiency of signal amplification but also enables primer generation to simultaneous cascade RCA amplification. Additionally, the electrochemical detection technology based on silver nanoclusters overcomes the shortcomings of traditional detection methods such as low sensitivity and complex operation. The detection limit achieved was 9.3 aM with a wide dynamic response ranging from 10 aM to 100 pM (at the DPV peak of - 0.5 V), which is comparable to most of the reported studies. Therefore, our work provided an ultra-sensitive way for the detection of miRNAs using nanostructures and revealed an effective means for disease theranostics and cancer diagnosis. In this work, for the first time, we designed a nucleic acid lock nanostructure based on its self-structural transformation for the specific detection of miRNA. And the combination of DNAzyme and cascade RCA reaction greatly improved the signal amplification efficiency.

Comparisons of urea or ammonium on growth and fermentative metabolism of Saccharomyces cerevisiae in ethanol fermentation.

This work was mainly about the understanding of how urea and ammonium affect growth, glucose consumption and ethanol production of S. cerevisiae, in particular regarding the basic physiology of cell. The basic physiology of cell included intracellular pH, ATP, NADH and enzyme activity. Results showed that fermentation time was reduced by 19% when using urea compared with ammonium. The maximal ethanol production rate using urea was 1.14 g/L/h, increasing 30% comparing with the medium prepared with ammonium. Moreover, urea could decrease the synthesis of glycerol from glucose by 26% comparing with ammonium. The by-product of acetic acid yields decreased from 40 mmol/mol of glucose (with urea) to 24 mmol/mol of glucose (with ammonium). At the end of ethanol fermentation, cell number and pH were greater with urea than ammonium. Comparing with urea, ammonium decreased the intracellular pH by 14% (from 7.1 to 6.1). Urease converting urea into ammonia resulted in a more than 50% lower of ATP when comparing with ammonium. The values of NADH/DCW were 0.21 mg/g and 0.14 mg/g respectively with urea and ammonium, suggesting a 33% lower NADH. The enzyme activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was 0.0225 and 0.0275 U/mg protein respectively with urea and ammonium, which was consistent with the yields of glycerol.

Toehold-mediated DNA strand displacement-driven super-fast tripedal DNA walker for ultrasensitive and label-free electrochemical detection of ochratoxin A.

DNA walkers, as intelligent artificial DNA nanomachines, have been widely used as efficient nucleic acid amplification tools that the detection sensitivity can be improved by incorporating DNA walkers into DNA biosensors. Nevertheless, since the premature release or flameout in a region of locally exhausted substrate, the walking efficiency of DNA walkers remains unsatisfactory. In this work, we design a smart tripedal DNA walker that is formed by target-initiated catalyzed hairpin assembly (CHA), which can move along the DNA duplex tracks on electrode driven by toehold-mediated DNA strand displacement (TMSD) for transduction and amplification of electrochemical signals. Emphatically, this flexible tripedal DNA walker is capable of walking freely along the tracks with unconstrained walking range. Moreover, the design of multi-legged walker can weaken the derailment of leg DNA and shorten the moving time on electrode, ensuring the processive walking with high efficiency. Additionally, the persistent walking of tripedal walker is driven by cascading TMSD, which eliminates the defects of high cost and instability of enzyme-assisted amplification technology. Therefore, the tripedal DNA walker-based electrochemical biosensor has enormous potential for the applications of OTA detection, and reveals a new avenue for food safety analysis and clinical diagnosis.

AKT/FOXO1 axis links cross-talking of endothelial cell and pericyte in TIE2-mutated venous malformations.

BACKGROUND: Venous malformations (VMs), most of which associated with activating mutations in the endothelial cells (ECs) tyrosine kinase receptor TIE2, are characterized by dilated and immature veins with scarce smooth muscle cells (SMCs) coverage. However, the underlying mechanism of interaction between ECs and SMCs responsible for VMs has not been fully understood. METHODS: Here, we screened 5 patients with TIE2-L914F mutation who were diagnosed with VMs by SNP sequencing, and we compared the expression of platelet-derived growth factor beta (PDGFB) and α-SMA in TIE2 mutant veins and normal veins by immunohistochemistry. In vitro, we generated TIE2-L914F-expressing human umbilical vein endothelial cells (HUVECs) and performed BrdU, CCK-8, transwell and tube formation experiments on none-transfected and transfected ECs. Then we investigated the effects of rapamycin (RAPA) on cellular characteristics. Next we established a co-culture system and investigated the role of AKT/FOXO1/PDGFB in regulating cross-talking of mutant ECs and SMCs. RESULTS: VMs with TIE2-L914F mutation showed lower expression of PDGFB and α-SMA than normal veins. TIE2 mutant ECs revealed enhanced cell viability and motility, and decreased tube formation, whereas these phenotypes could be reversed by rapamycin. Mechanically, RAPA ameliorated the physiological function of mutant ECs by inhibiting AKT-mTOR pathway, but also facilitated the nuclear location of FOXO1 and the expression of PDGFB in mutant ECs, and then improved paracrine interactions between ECs and SMCs. Moreover, TIE2 mutant ECs strongly accelerated the transition of SMCs from contractile phenotype to synthetic phenotype, whereas RAPA could prevent the phenotype transition of SMCs. CONCLUSIONS: Our data demonstrate a previously unknown mechanistic linkage of AKT-mTOR/FOXO1 pathway between mutant ECs and SMCs in modulating venous dysmorphogenesis, and AKT/FOXO1 axis might be a potential therapeutic target for the recovery of TIE2-mutation causing VMs. Video Abstract.

Highly efficient fluorescence sensing of kanamycin using Endo IV-powered DNA walker and hybridization chain reaction amplification.

An ultrasensitive fluorescence sensing strategy for kanamycin (KANA) determination using endonuclease IV (Endo IV)-powered DNA walker, and hybridization chain reaction (HCR) amplification was reported. The sensing system consists of Endo IV-powered 3D DNA walker using for the specific recognition of KANA and the formation of the initiators, two metastable hairpin probes as the substrates of HCR and a tetrahydrofuran abasic site (AP site)-embeded fluorescence-quenched probe for fluorescence signal output. On account of this skilled design of sensing system, the specific binding between KANA and its aptamer activates DNA walker, in which the swing arm can move autonomously along the 3D track via Endo IV-mediated hydrolysis of the anchorages, inducing the formation of initiators that initiates HCR and the following Endo IV-assisted cyclic cleavage of fluorescence reporter probes. The use of Endo IV offers the advantages of simplified and accessible design without the need of specific sequence in DNA substrates. Under the optimal experimental conditions, the fluorescence biosensor shows excellent sensitivity toward KANA detection with a detection limit as low as 1.01 pM (the excitation wavelength is 486 nm). The practical applicability of this strategy is demonstrated by detecting KANA in spiked milk samples with recovery in the range of 98 to 102%. Therefore, this reported strategy might create an accurate and robust fluorescence sensing platform for trace amounts of antibiotic residues determination and related safety analysis. Graphical abstract Highly efficient fluorescence sensing of kanamycin using Endo IV-powered DNA Walker and hybridization chain, reaction amplification, Xiaonan Qu, Jingfeng Wang, Rufeng Zhang, Yihan Zhao, Shasha Li, Yu Wang, Su Liu*, Jiadong Huang, and Jinghua Yu, an ultrasensitive fluorescence sensing strategy for kanamycin determination using endonuclease IV-powered DNA walker, and hybridization chain reaction amplification is reported.

Efficient strand displacement amplification via stepwise movement of a bipedal DNA walker on an electrode surface for ultrasensitive detection of antibiotics.

DNA walkers, one of the artificial molecular machines which are constructed via smart synthetic DNA, have attracted rapidly growing attention from researchers in the biosensing field. In this work, we design an Exonuclease III (Exo III)-aided target-aptamer binding recycling (ETBR) activated bipedal DNA machine for highly sensitive electrochemical detection of antibiotics. To the best of our knowledge, this is the first time that a bipedal DNA machine has been applied in electrochemical sensing for antibiotics. On the one hand, the bipedal DNA walker exceeds the conventional single swing arm DNA walker in terms of walking efficiency and stability. On the other hand, the ETBR strategy, along with efficient strand displacement amplification via stepwise movement of a bipedal DNA walker significantly promotes the signal amplification efficiency. Under optimal conditions, this bipedal DNA machine possesses a detection limit of 7.1 fM within a linear detection range from 10 fM to 100 pM. Moreover, this electrochemical biosensor is expected to detect a wide variety of analytes using the corresponding target recognition probes. Thus, our proposed strategy offers a highly efficient, stable and practical platform for small molecule analysis.