An Ultramicroelectrode Electrochemistry and Surface Plasmon Resonance Coupling Method for Cell Exocytosis Study.

Exocytosis of a single cell has been extensively researched in recent years due to its close association with numerous diseases. However, current methods only investigate exocytosis at either the single-cell or multiple-cell level, and a method for simultaneously studying exocytosis at both levels has yet to be established. In this study, a combined device incorporating ultramicroelectrode (UME) electrochemistry and surface plasmon resonance (SPR) was developed, enabling the simultaneous monitoring of single-cell and multiple-cell exocytosis. PC12 cells were cultured directly on the SPR sensing Au film, with a carboxylated carbon nanopipette (c-CNP) electrode employed for electrochemical detection in the SPR reaction cell. Upon exocytosis, the released dopamine diffuses onto the inner wall of c-CNP, undergoing an electrochemical reaction to generate a current peak. Concurrently, exocytosis can also induce changes in the refractive index of the Au film surface, leading to the SPR signal. Consequently, the device enables real-time monitoring of exocytosis from both single and multiple cells with a high spatiotemporal resolution. The c-CNP electrode exhibited excellent resistance to protein contamination, high sensitivity for dopamine detection, and the capability to continuously monitor dopamine exocytosis over an extended period. Analysis of both SPR and electrochemical signals revealed a positive correlation between changes in the SPR signal and the frequency of exocytosis. This study introduces a novel method and platform for the simultaneous investigation of single-cell and multiple-cell exocytosis.

Atomically dispersed Au anchored on CeO2to enhancing the antioxidant activity.

The modification of Au nanoparticles can improve the antioxidant activity of CeO2, however, nano Au/CeO2has also met some problems such as low atomic utilization, the limit of reaction conditions, and high cost. Au single atom catalysts can well solve the above-mentioned problems, but there are some contradictory results about the activity of single atom Au1/CeO2and nano Au/CeO2. Here, we synthesized rod-like Au single atom Au/CeO2(0.4% Au1/CeO2) and nano Au/CeO2(1% Au/CeO2, 2% Au/CeO2and 4% Au/CeO2), and their antioxidant activity from strong to weak is 0.4% Au1/CeO2, 1% Au/CeO2, 2% Au/CeO2and 4% Au/CeO2, respectively. The higher antioxidant activity of 0.4% Au1/CeO2is mainly due to the high Au atomic utilization ratio and the stronger charge transfer between Au single atoms and CeO2, resulting in the higher content of Ce3+. Due to the coexistence of Au single atoms and Au NPs in 2% Au/CeO2, the antioxidant activity 2% Au/CeO2is higher than that of 4% Au/CeO2. And the enhancement effect of Au single atoms was not affected by the concentration of ·OH and material concentration. These results can promote the understanding of the antioxidant activity of 0.4% Au1/CeO2and promote its application.

The transcript NR 134251.1 of lncRNA APTR with an opposite function to all transcripts inhibits proliferation and induces apoptosis by regulating proliferation and apoptosis-related genes.

Arsenic (As) exposure has been a global public health concern for hundreds of millions worldwide. LncRNA APTR (Alu-mediated p21 transcriptional regulator) plays an essential role in tumor growth and development. However, its function in arsenic-induced toxicological responses is still unknown. In this study, we found that the expressions of all transcripts and the transcript NR 134251.1 of APTR were increased in a dose-dependent manner in 16HBE cells treated with sodium arsenite (NaAsO2). Silencing the transcript NR 134251.1 of APTR inhibited cell proliferation and induced apoptosis. However, silencing all transcripts of APTR had the opposite function to the transcript NR 134251.1. Then we examined the protein level of the proliferation and apoptosis-related genes after silencing the transcript NR 134251.1 of APTR. The results showed that silencing the transcript NR 134251.1 of APTR up-regulated the expression of transcription factor E2F1 and regulated its downstream genes involved in proliferation and apoptosis, including p53, phospho-p53-S392, phospho-p53-T55, p21, Cyclin D1, PUMA, Fas, Bim, BIK, Caspase-3, Caspase-7, and Cyt-c. In conclusion, arsenic induced APTR expression and the transcript NR 134251.1 of APTR have an opposite function to all transcripts, providing a theoretical basis for the prevention and treatment of arsenic exposure.

A simple method for the preparation of CeO2with high antioxidant activity and wide application range.

Cerium oxide (CeO2) is a well-known antioxidant with the ability to scavenge reactive oxygen species due to its unique electronic structure and chemical properties. Although many methods to enhance the antioxidant activity of CeO2have been reported, its antioxidant activity is still not high enough, and some enhancement effects are limited by the material concentration. There are also some CeO2obtained with high antioxidant activity at high concentrations, which is not conducive to the application of biomedicine. Therefore, it is urgent to obtain CeO2material with low cell cytotoxicity, high antioxidant activity and wide application range. In this work, rod-like metal organic framework derived CeO2(CeO2-MOF) was prepared by a simple method. Compared with the CeO2nanorods prepared by hydrothermal method, it shows better antioxidant activity compared with the CeO2nanorods prepared by hydrothermal method. Moreover, the advantage of CeO2-MOF's antioxidant activity is not affected by the hydroxyl radical and material concentrations The reason why CeO2-MOF has higher antioxidant activity should be attributed to its higher Ce3+content and larger specific surface area. In addition, CeO2-MOF also exhibits low cytotoxicity to HeLa cells and PC12 cellsin vitro. The strategy of using MOF as a structural and compositional material to create CeO2provides a new method to explore highly efficient and biocompatible CeO2for practical applications.

Hsa_circ_0005050 interacts with ILF3 and affects cell apoptosis and proliferation by disrupting the balance between p53 and p65.

The regulatory network between arsenic, genes and signaling pathways has been reported in arsenic carcinogenesis. Studies on circRNA represent a growing field, but the extent to circRNA potential mechanisms remains poorly understood. So this study we explore the systematic function of hsa_circ_0005050 in mediating the cell apoptosis and proliferation. We demonstrated that hsa_circ_0005050 was highly expressed in subjects who are long-term exposed to arsenic, and could be induced by NaAs2O3 in A549 and 16HBE. Knockdown of hsa_circ_0005050 promotes A549 cell viability, whereas exerts the opposite effects in 16HBE. Mechanistically, hsa_circ_0005050 regulates the p53 and NF-κB signaling pathway involved in the apoptosis and proliferation. And we found that hsa_circ_0005050 could directly bind to the RNA binding protein ILF3 and mutually influence each other's formation. Upon si-hsa_circ_0005050, ILF3 export to the cytoplasm resulting the formation of a ternary complex ILF3-p65-IκBA, breaks the balance of p53 and NF-κB pathway and induces A549 apoptosis and leads to 16HBE proliferation. As a result of these investigations, suggestions were identified for future research.

CircP50 functions through the phosphorylation- and acetylation-activated p53 pathway to mediate inorganic arsenic-induced apoptosis in A549 cells.

As a class I carcinogen, arsenic has been reported to cause diseases accompanied by circRNAs regulating proliferation and apoptosis at the molecular level, but whether circP50 (circBase ID: hsa_circ_0008012) does the same has not been demonstrated. The aim of this study is to provide the basis for anti-lung cancer mechanism research, by studying the expression of circP50 under arsenic-induced conditions, and the effect and mechanism on the proliferation and apoptosis of A549 cells based on the circP50 knockdown models. To explore whether the circP50 is responsive to arsenic exposure, the qRT-PCR was applied to discover that the relative expression of circP50 in A549 cells increased only with increasing NaAsO2 dose and independent of its metabolites. We further determined the mechanism of circP50 by establishing circP50 knockdown models. The results of cell viability and EdU assays indicated the proliferation of A549 cells. According to the western blotting, phosphorylation of p53 at Ser15, Ser376, and Ser392 and acetylation of p53 at Lys370 and Lys382 were inhibited, resulting in the deficiency of p53 expression. Subsequently, the expression of genes downstream of p53 was reduced, including p21, PUMA, Caspase3, and Bcl-xS. Furthermore, the expressions of IKB-α, p65, and p50 decreased, but C-myc expression did not change significantly, referring to the NF-κB pathway was not dominant. The results suggest that circP50 mainly functions through the p53 pathway to mediate apoptosis in response to arsenic exposure.

A lable-free SPR biosensor based on one peptide sequence with three recognition sites for O-GlcNAc transferase detection.

Abnormal O-linked N-acetylglucosamine (O-GlcNAc) concentrations have been associated with many diseases, but the lack of accurate detection method limited O-GlcNAc to be used as a biomarker in clinical diagnosis. Then O-GlcNAc transferase (OGT) has drawn researchers' attention as it closed related to the level of O-GlcNAc and be considered to be a promising new target for diseases diagnosis. Nevertheless, the existing OGT detection methods are either need labeling or the sensitity can not meet the needs of clinic testing. Herein, a label-free and sensitive SPR biosensor was developed for accurate detection of OGT based on a multi-functional peptide. The designed peptide contains three recognition sites, one is the cleavage site of protease K, one is the O-GlcNAcylated site by OGT, and another is six histidine which be used as the signal report probe to recognize Ni2+. The immobilized peptide would be cleavaged by proteinase K, then the His-tag residue part will leave the surface of Au film, resulting less His-tag could bind to Ni2+ and a small SPR signal would be record. If the peptide is O-GlcNAcylated by OGT, the cleaving reaction would be limited due to the adjacent site of O-GlcNAcylation. Then more His-tag can be left on the Au film and a bigger SPR signal could be record, this signal is associated with the concentration of OGT. Utilizing the change of the peptide configuration as a signal report probe for OGT detection not only avoids labeling of peptide, but also makes the method more sensitive. The determination linear range of OGT is from 2.00 × 10-13 to 5.00 × 10-8 M with a detection limit of 1.19 × 10-13 M, and the separation of two enzyme reactions ensured the high selectivity of the method. Finally, the sensing system was successfully used for OGT detection in blood samples with satisfied recovery. In summary, the label-free SPR platform for accurate detection of OGT in real samples is helpful to promote OGT serve as a biomarker for early clinical diagnosis of O-GlcNAc related diseases.

Enhancement of antioxidant activity of well-dispersed core-shell structured CeO2 coating Au nanorods under visible light irradiation.

Gold nanoparticle (AuNP) modification shows great advantages in improving the antioxidant activity of nanoCeO2. However, the improved effect of AuNP modification becomes smaller and even results in the decrease of antioxidant ability due to severe aggregation with increasing nanomaterial concentration. Additionally, the effects of photo-properties of AuNPs on the antioxidant activity of nanoCeO2 have not been studied. In response to these problems, core-shell-shaped Au@CeO2 was synthesized which took Au nanorods (AuNRs) as carriers and had a layer of CeO2 NP coating. The antioxidant activity of Au@CeO2 was evaluated by the UV-vis method in the methyl violet-Fenton system. Results showed that AuNRs could improve the antioxidant activity of nanoCeO2 due to the increase in the amount of Ce3+ on the surface of nanoCeO2, and the enhancing effect remained across the whole experimental concentration range due to the good dispersibility of AuNRs. Additionally, a further increase in the antioxidant ability of Au@CeO2 was found with 5 min visible light irradiation, and continuous irradiation during a 25 min time reaction, which resulted in more obvious enhanced antioxidant ability. This phenomenon was attributed to the localized surface plasmon resonance of AuNRs triggered by photons which induced charge transfer from AuNRs to nanoCeO2, thus making the cyclic transformation between Ce3+ and Ce4+ easier.

Surface plasmon resonance biosensor for the accurate and sensitive quantification of O-GlcNAc based on cleavage by ß-D-N-acetylglucosaminidase.

Abnormal O-linked-N-acetylglucosamine (O-GlcNAc) concentrations have been associated with a variety of diseases (e.g., cancer, Alzheimer's disease, cardiovascular disease, etc.). However, O-GlcNAc detection is complicated, time-consuming and has poor specificity, therefore, the accurate detection of O-GlcNAc is difficult. In this study, an accurate and sensitive surface plasmon resonance (SPR) biosensor for O-GlcNAc detection that is based on ß-D-N-acetylglucosaminidase (OGA) and Au nanoparticles (AuNPs) was developed. In this strategy, AuNPs were used to amplify the SPR signal and improve the biosensor's sensitivity; OGA was used to cleave O-GlcNAc from O-GlcNAcylated biomolecules. The interaction between AuNPs labeled wheat germ agglutinin (AuNPs/WGA) and O-GlcNAcylated biomolecules on a modified Au film treated with and without OGA was recorded by SPR. The change of the SPR signal moves linearly with the amount of O-GlcNAc on the Au film and thus could be used for the detection of O-GlcNAc. By recording the difference of the SPR signals, this method can avoid disturbances from other sugars and nonspecific adsorption of AuNPs and thus enable the accurate detection of O-GlcNAc. The accurate detection range of O-GlcNAc was 4.65 × 10-12 to 4.65 × 10-7 M which was obtained by quantifying the amount of a standard O-GlcNAcylated peptide (O-GlcNAc-CREB), and the detection limit is 4.65 × 10-13 M. More importantly, the strategy was successfully used to detect O-GlcNAc in a real α-crystallin protein, cancer cell lysates and blood samples with satisfactory results. The study's results imply that this accurate and sensitive method has the potential to be applied in the early clinical diagnosis of O-GlcNAc-related diseases.

The effect of DNA on the oxidase activity of nanoceria with different morphologies.

Many nanomaterials have been reported to have enzyme-like activities and are considered as nanozymes. As a multifunctional nanozyme, nanoceria has received much attention due to the dual oxidation states of Ce3+/Ce4+ which facilitate redox reactions at the particle surface. Despite the advantages of nanozymes, their limited activity and lack of enzyme specificity are still problems to be resolved. DNA is used to modulate the oxidase activity of nanoceria because it has recently become an important molecule in bionanotechnology. However, the current research on the effect of DNA on the oxidase mimetic activity of nanoceria is contradictory. It has been discovered that nanoceria used in recent works are different, including in particle size, doping and concentration, and these differences may affect the interaction between DNA and nanoceria, and then affect the oxidase mimetic activity of nanoceria. Hence, it is important to clarify the factors that affect the interaction between DNA with nanoceria. In this work, the interactions between DNA and nanoceria with three different morphologies (nanoparticles, nanocubes, and nanorods) have been investigated. Experimental results show that DNA has different influences on the oxidase mimetic activity of nanoceria with different morphologies. The oxidase mimetic activity of CeO2 nanoparticles and nanocubes increased, but that of CeO2 nanorods decreased, after DNA modification. The mechanism of these experimental results has been explored, and it has been found that it is the interaction between cerium and the phosphate backbone of DNA that changes with the different morphologies, resulting in the varying effect of DNA on the oxidase mimetic activity of nanoceria. These results may provide a better understanding of the effect of DNA on the oxidase mimetic activity of nanoceria and promote the applications of nanoceria.

[Application of adjustable moving overlapping separation range map in multisegment stepwise gradient elution condition optimization of high performance liquid chromatography].

Computer-assisted optimization of high performance liquid chromatographic separation conditions can be used to obtain optimized experimental conditions in relatively short time and low cost, and has been widely applied to the separation of complex samples. Based on "moving overlapping separation range map" method, a novel method of optimizing multi-segment stepwise gradient elution conditions, "adjustable moving overlapping separation range map" method, was developed. By predicting the retention time, peak width and resolution of each solute under different mobile phase conditions, the overlapping separation range map of the solutes in the sample is plotted. When the mobile phase composition on the current step is optimized, the overlapping separation range map for solutes eluted in next one or two steps is replotted simultaneously by considering the influence of elution conditions for current step on the retention of uneluted solutes. It is helpful to consider the separation of each solute in the sample simultaneously and find optimized separation conditions close to the global optimum by investigating the effect of mobile phase conditions for current step on the separation of solutes eluted in the current and next one or two steps. By slightly adjusting the optimized separation conditions using grid search method, the separation performance can be further improved. The application of "adjustable moving overlapping separation range map" method was illustrated by using experimental data from literature. The advantage of this method in high performance liquid chromatography was verified in binary mobile phase system.

Separation and identification of compounds in Rhizoma chuanxiong by comprehensive two-dimensional liquid chromatography coupled to mass spectrometry.

A comprehensive two-dimensional liquid chromatographic separation system based on the combination of a CN column and an ODS column is developed for the separation of components in a traditional Chinese medicine (TCM) Rhizoma chuanxiong. Two columns are coupled by a two-position, eight-port valve equipped with two storage loops and controlled by a computer. The effluent is detected by both the diode array detector and atmospheric pressure chemical ionization (APCI) mass spectrometer. More than 52 components in the methanol extract of R. chuanxiong were resolved and 11 of them were preliminary identified according to their UV and mass spectra.

[Fast optimization of stepwise gradient conditions for ternary mobile phase in reversed-phase high performance liquid chromatography].

In high performance liquid chromatography, it is necessary to apply multi-composition gradient elution for the separation of complex samples such as environmental and biological samples. Multivariate stepwise gradient elution is one of the most efficient elution modes, because it combines the high selectivity of multi-composition mobile phase and shorter analysis time of gradient elution. In practical separations, the separation selectivity of samples can be effectively adjusted by using ternary mobile phase. For the optimization of these parameters, the retention equation of samples must be obtained at first. Traditionally, several isocratic experiments are used to get the retention equation of solute. However, it is time consuming especially for the separation of complex samples with a wide range of polarity. A new method for the fast optimization of ternary stepwise gradient elution was proposed based on the migration rule of solute in column. First, the coefficients of retention equation of solute are obtained by running several linear gradient experiments, then the optimal separation conditions are searched according to the hierarchical chromatography response function which acts as the optimization criterion. For each kind of organic modifier, two initial linear gradient experiments are used to obtain the primary coefficients of retention equation of each solute. For ternary mobile phase, only four linear gradient runs are needed to get the coefficients of retention equation. Then the retention times of solutes under arbitrary mobile phase composition can be predicted. The initial optimal mobile phase composition is obtained by resolution mapping for all of the solutes. A hierarchical chromatography response function is used to evaluate the separation efficiencies and search the optimal elution conditions. In subsequent optimization, the migrating distance of solute in the column is considered to decide the mobile phase composition and sustaining time of the latter steps until all the solutes are eluted out. Thus the first stepwise gradient elution conditions are predicted. If the resolution of samples under the predicted optimal separation conditions is satisfactory, the optimization procedure is stopped; otherwise, the coefficients of retention equation are adjusted according to the experimental results under the previously predicted elution conditions. Then the new stepwise gradient elution conditions are predicted repeatedly until satisfactory resolution is obtained. Normally, the satisfactory separation conditions can be found only after six experiments by using the proposed method. In comparison with the traditional optimization method, the time needed to finish the optimization procedure can be greatly reduced. The method has been validated by its application to the separation of several samples such as amino acid derivatives, aromatic amines, in which satisfactory separations were obtained with predicted resolution.

[Application of genetic algorithm in optimization of mobile phase composition in high performance liquid chromatography].

Referring to traditional optimal methods, a method for the optimization of isocratic elution mobile phase composition in high performance liquid chromatography has been developed. In this method, the genetic algorithm based on line-crossover and plane-mutation is used. The principle of genetic algorithm and the process of optimization of mobile phase composition in reversed-phase ion-pair high performance liquid chromatography using genetic algorithm are introduced in details. With the concentrations of acetonitrile and ion-pair reagent sodium octane sulfonate chosen as the optimal parameters, the optimum was obtained by three times of optimization procedures. The mean relative error between the predicted and experimental values was 0.75% at the optimum and the optimization results were satisfactory.