Evaluation of biological activity of an eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody / 中国生物制品学杂志

@#Objective To evaluate the biological activity of a eukaryotic expressed anti-H5N1-M1 cell entry single molecule antibody(TAT-ScFv-mFc). Methods The immune binding activity and affinity of TAT-ScFv-mFc to H5N1-M1 protein were detected by Western blot and localized surface plasmon resonance(LSPR)respectively;The inhibitory effect of TAT-ScFv-mFc on influenza virus H1N1 was detected by CCK-8 assay;The membrane penetration ability of TAT-ScFv-mFc to MDCK cells was verified by immunofluorescence assay. A total of 30 female BALB/c mice were injected with TAT-ScFv-mFc via tail vein,200 μL per mouse. Blood samples were collected at 5,60,120,240 and 360 min after injection. Serum samples were separated and detected for the titers by ELISA,and the half-life of TAT-ScFv-mFc was calculated according to the half-life curve drawn by Origin 2021 software. Results TAT-ScFv-mFc showed specific binding to H5N1-M1 protein with a binding rate constant of 6. 67 × 10~4[1/（M*s）]. The survival rate of MDCK cells infected by H1N1 increased gradually with the increase of TAT-ScFv-mFc concentration in a dose-dependent manner,which obviously inhibited the replication of H1N1. TAT-ScFv-mFc penetrated the cell membrane of MDCK cells in a short time,entered the cell and bound to virus M1protein,thus inhibiting virus replication and assembly. The half-life of TAT-ScFv-mFc in mice was 212 min. Conclusion TAT-ScFv-mFc has good immune binding activity and affinity with H5N1-M1,can effectively inhibit the replication of H1N1,has good penetration ability to MDCK cell membrane,and has a long half-life in mice,which lays a foundation of the drug treatment,vaccine research and preventive treatment of H5N1 infection.

Characteristic intestinal flora and associated biomarkers in esophageal cancer based on PacBio SMRT sequencing / 环境与职业医学

Background Esophageal cancer is a common gastrointestinal tumor with a high incidence in China. Some studies suggest that intestinal flora is significantly related to the occurrence and development of tumors and other diseases. Traditional 16S rDNA sequencing technology only provides taxonomic resolution at genus level. Objective Based on PacBio single molecule real time (SMRT) sequencing technology to identify characteristic microbial biomarkers associated with esophageal cancer at the species level. Methods A total of 120 newly diagnosed cases of esophageal cancer were recruited and 60 healthy patients with matched sex and age were recruited as the control group. Fresh stool samples were collected from all subjects. Full-length 16S rDNA sequencing was performed on samples from 4 patients with esophageal cancer and 1:1 matched healthy controls using the third-generation sequencing PacBio SMRT technology, and the structural differences of intestinal flora were analyzed based on the sequencing results. Function prediction was performed by PICRUSt software. Large population samples were validated by screening different gut microbes by linear discriminant analysis and linear discriminant analysis effect size to identify esophageal cancer-associated gut microbes. Results Based on sequencing samples, the results of α diversity analysis showed that the Ace, Chao1, Simpson Diversity, and Shannon Wiener indices of the esophageal cancer group were higher than those of the healthy control group (P<0.05), and the results of β diversity showed that the scattered clusters of the esophageal cancer group and the healthy control group were separated, which meant that there were differences in the structure of intestinal flora between the two groups. It was found at the phylum level that the abundances of Proteobacteria, Bacteroidetes, and Firmicutes in the intestinal flora of the esophageal cancer group were increased. At the genus level, the relative abundances of Spirospira, Pasteurella, Roxella, and Bacteroides in the esophageal cancer group were increased. At the species level, there were 11 microbial species with increased relative abundances in the esophageal cancer group, including Enterobacter sp. E.20, Bacteroides ovatus V975, and Faecalibacterium prausnitzii, and the microbial species with decreased relative abundances in the esophageal cancer group were Ralstonia pickettii, Enterobacter unclassified, and Streptococcus salivarius JIM8777. The PICRUSt functional annotation found differences in alanine, aspartate and glutamate metabolism (map00250), peptidoglycan (map00550), one carbon pool by folate (map00670), thiamine metabolism (map00730), and biosynthesis of amino acids (map01230) between the two groups. The results of the population validation study showed that the abundances of Enterobacter sp E.20 and Bacteroides massilience in the esophageal cancer group were increased, the abundance of Streptococcus salivarius JIM8777 was decreased, and the differences between the two groups were statistically significant (P<0.05). By establishing receiver operating characteristic analysis for representative species level biomarkers, the area under curve (AUC) of combining Enterobacter sp E.20, Streptococcus salivarius JIM8777, and Bacteroides massilience was 0.779, higher than single diagnosis (AUC=0.610, 0.608, and 0.659, respectively). Conclusion There are significant differences in gut microbiota between the esophageal cancer group and the healthy control group. The combination of Enterobacter sp E.20, Streptococcus salivarius JIM8777, and Bacteroides Massilience has potential application value for the diagnosis of esophageal cancer.

Research progress on single-molecule array technology-based blood neuronal markers for Alzheimer's disease / 中华老年医学杂志

Early detection and intervention is an important strategy for the prevention and treatment of Alzheimer's disease(AD). Blood screening for AD has advantages such as low cost and convenient sample collection.However, there are also problems such as low levels of markers in peripheral blood and insufficient sensitivity of detection methods.At present, as a highly sensitive detection technology for protein molecules, single-molecule array(Simoa)technology can accurately analyze low-concentration biomarkers that cannot be detected by traditional methods.This article reviews research progress on blood neuronal markers for early AD diagnosis based on the new Simoa technology.

Using PacBio sequencing to investigate the effects of treatment with lactic acid bacteria or antibiotics on cow endometritis

BACKGROUND: Endometritis is the most common disease of dairy cows and traditionally treated with antibiotics. Lactic acid bacteria can inhibit the growth of pathogens and also have potential for treatment of endometritis. Using PacBio single-molecule real-time sequencing technology, we sequenced the fulllength l6S rRNA of the microbiota in uterine mucus samples from 31 cows with endometritis, treated with lactic acid bacteria (experimental [E] group) and antibiotics (control [C] group) separately. Microbiota profiles taken before and after treatment were compared. RESULTS: After both treatments, bacterial species richness was significantly higher than before, but there was no significant difference in bacterial diversity. Abundance of some bacteria increased after both lactic acid bacteria and antibiotic treatment: Lactobacillus helveticus, Lactococcus lactis, Lactococcus raffinolactis, Pseudomonas alcaligenes and Pseudomonas veronii. The bacterial species that significantly decreased in abundance varied depending on whether the cows had been treated with lactic acid bacteria or antibiotics. Abundance of Staphylococcus equorum and Treponema brennaborense increased after lactic acid bacteria treatment but decreased after antibiotic treatment. According to COG-based functional metagenomic predictions, 384 functional proteins were significantly differently expressed after treatment. E and C group protein expression pathways were significantly higher than before treatment (p < 0.05). CONCLUSIONS: In this study, we found that lactic acid bacteria could cure endometritis and restore a normal physiological state, while avoiding the disadvantages of antibiotic treatment, such as the reductions in abundance of beneficial microbiota. This suggests that lactic acid bacteria treatment has potential as an alternative to antibiotics in the treatment of endometritis in cattle.

Application of SM-PCR to detect plasma ctDNA in the treatment of patients with ad-vanced lung adenocarcinoma / 中国肿瘤临床

Objective: To investigate the application of single-molecule PCR (SM-PCR) in the detection of plasma ctDNA for the treat-ment of patients with advanced lung adenocarcinoma. Methods: In total, 30 patients diagnosed with advanced lung adenocarcinoma were enrolled between June 2017 and May 2018. ctDNA fragments of the target genes (EGFR, KRAS, BRAF, ALK, HER2, and TP53) from the blood samples were enriched by SM-PCR, and DNA libraries were prepared. Finally, a high-throughput sequencing was performed. The EGFR detection of tumor tissue samples was performed using real-time fluorescence PCR based on the amplification refractory mutation system (ARMS) and consistency in the results of EGFR mutation detection in the plasma and tissue was compared. Results:The results of both the methods were consistent (Kappa=0.867, P<0.001). The McNemar's test also indicated that the results are not statistically different (P=0.500). Conclusions: SM-PCR can be used for the detection of plasma EGFR mutations. The target detection sites are more comprehensive and multiple mutations can be detected at the same time. Results of the analysis are more precise and can be absolutely quantified.

Single-molecule methods in study of G protein-coupled receptor dimmers / 中国药理学通报

G-protein coupled receptors (GPCRs) can form biologically relevant dimers and oligomers, which are important to regulate receptor function. They have specific signal transduction pathways, thus mediating a series of physiological and pathological processes, such as cardiovascular regulation, energy metabolism, etc. GPCR dimers become one of the new drug targets, attracting increasing attention. Traditionally, the research on GPCR dimers have been performed on a population " ensemble average" level. However, the main problem is that there may be valuable information hidden in this " noise". Single molecule technology has unprecedented spatial and temporal resolution, which can directly display the internal state, trajectory of GPCR dimers and the changes with time and surrounding environment. It helps to further analyze the key role and drug targets of GPCR dimers. Here, we review single-molecule techniques (such as total internal reflection fluorescence microscopy, stimulated emis-sion loss microscopy and ground state depletion microscopy, etc.) that have been applied to GPCR dimers.

Progress in Solid-state Nanopore-based Analysis Technology / 分析化学

Nanopore technique is a low-cost tool for single-molecule level analysis without the need of label or amplification. The solid nanopores have been widely used in many fields such as chemistry and life sciences due to their advantages such as high stability, good tolerability, controllable size, and easy for modification. The solid nanopores commonly used in recent years are fabricated using two types of materials: membrane and tube. The membrane materials include silicon nitride, two-dimensional materials, aluminium oxide, and polymer membranes. The tube materials mainly include glass capillary and carbon nanotube. This review summarizes and prospects the research progress of different solid nanopores.

Applications of Nanopore Sensing in Detection of Toxic Molecules / 分析化学

As a novel analytical method, nanopore sensing is widely applied in many fields such as nucleic acids sequencing, protein / peptide analysis, detection of metal ions and biomacromolecules including virus, bacteria, etc. With the growing public concerns over dietary safety and public security, there has been a greater demand on the detection of toxic molecules. With its high sensitivity and selectivity, nanopore sensing is considered as a more powerful assay, which has been reported in many research articles. Accordingly, this paper surveys the application studies of nanopore sensing in detection of toxic molecules.

Application of atomic force microscopy-based single molecule force spectroscopy in G-quadruplex studies / 南方医科大学学报

Telomere plays a crucial role in the physiological and pathological processes of cells. At the end of the telomere, the single-stranded DNA repeat sequence rich in guanine (G) folds in the presence of monovalent metal ions such as Na or K to form a G-quadruplex structure. This structure can not be extended by telomerase and inhibits the activity of telomerase, thus becoming a potential anticancer target. Stabilizing the formation of DNA G-quadruplex structures by small molecule ligands has become a new strategy for designing many anticancer drugs, and studying the interaction strength of these small molecule ligands with G-quadruplex is thus of particular importance for screening highly effective anticancer drugs. Single molecule force spectroscopy enables direct measurement of the interaction between small molecule ligands and G-quadruplexes. This review highlights the advances of single-molecule force spectroscopy based on atomic force microscopy in the study of the G quadruplex structure and its interaction with small molecule ligands, and summarizes the application and development trend of single molecule force spectrum technology in G quadruplex.

Biomonitoring for traditional herbal medicinal products using DNA metabarcoding and single molecule, real-time sequencing

Global concerns have been paid to the potential hazard of traditional herbal medicinal products (THMPs). Substandard and counterfeit THMPs, including traditional Chinese patent medicine, health foods, dietary supplements, etc. are potential threats to public health. Recent marketplace studies using DNA barcoding have determined that the current quality control methods are not sufficient for ensuring the presence of authentic herbal ingredients and detection of contaminants/adulterants. An efficient biomonitoring method for THMPs is of great needed. Herein, metabarcoding and single-molecule, real-time (SMRT) sequencing were used to detect the multiple ingredients in Jiuwei Qianghuo Wan (JWQHW), a classical herbal prescription widely used in China for the last 800 years. Reference experimental mixtures and commercial JWQHW products from the marketplace were used to confirm the method. Successful SMRT sequencing results recovered 5416 and 4342 circular-consensus sequencing (CCS) reads belonging to the ITS2 and regions. The results suggest that with the combination of metabarcoding and SMRT sequencing, it is repeatable, reliable, and sensitive enough to detect species in the THMPs, and the error in SMRT sequencing did not affect the ability to identify multiple prescribed species and several adulterants/contaminants. It has the potential for becoming a valuable tool for the biomonitoring of multi-ingredient THMPs.

Single Molecule Method for Molecular Biology

In order to understand biological phenomena accurately, single molecule techniques using a physical research approach to molecular interactions have been developed, and are now widely being used to study complex biological processes. In this review, we discuss some of the single molecule methods which are composed of two major parts: single molecule spectroscopy and manipulation. In particular, we explain how these techniques work and introduce the current research which uses them. Finally, we present the oral biology research using the single molecule methods.

Rapid and Accurate Sequencing of Enterovirus Genomes Using MinION Nanopore Sequencer / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>Knowledge of an enterovirus genome sequence is very important in epidemiological investigation to identify transmission patterns and ascertain the extent of an outbreak. The MinION sequencer is increasingly used to sequence various viral pathogens in many clinical situations because of its long reads, portability, real-time accessibility of sequenced data, and very low initial costs. However, information is lacking on MinION sequencing of enterovirus genomes.</p><p><b>METHODS</b>In this proof-of-concept study using Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) strains as examples, we established an amplicon-based whole genome sequencing method using MinION. We explored the accuracy, minimum sequencing time, discrimination and high-throughput sequencing ability of MinION, and compared its performance with Sanger sequencing.</p><p><b>RESULTS</b>Within the first minute (min) of sequencing, the accuracy of MinION was 98.5% for the single EV71 strain and 94.12%-97.33% for 10 genetically-related CA16 strains. In as little as 14 min, 99% identity was reached for the single EV71 strain, and in 17 min (on average), 99% identity was achieved for 10 CA16 strains in a single run.</p><p><b>CONCLUSION</b>MinION is suitable for whole genome sequencing of enteroviruses with sufficient accuracy and fine discrimination and has the potential as a fast, reliable and convenient method for routine use.</p>

Development of Biological Nanopore Technique in Non-gene Sequencing Application / 分析化学

Nanopore technique is a low-cost, ultrafast method for single-molecule level analysis without labels. Nanopore technique was first proposed more than 20 years ago and exhibited an excellent potential in DNA sequencing. So far, the commercial development of nanopore strand-sequencing as a portable device has been realized. Meanwhile, a remarkable number of studies have demonstrated that nanopore represents versatile single-molecule sensors for a wide range of molecule. Therefore, in this article we mainly review the use of nanopore technique based on the interface interactions between biological pore and the analytes such as protein/peptide to obtain kinetic and thermodynamic information at single-molecule level. And a large number of biological molecules and metal ions are quantitatively detected by nanopore analysis, allowing its development for the future biotechnologies and medicine applications. Besides, electrochemical detection system is crucial to nanopore technique. Therefore, we focus on advancements in relative software and ultralow current instrumentations with high-bandwidth.

Development of Integrated Atomic Force Microscopy and Fluorescence Microscopy for Single-Molecule Analysis in Living Cells / 分析化学

Atomic force microscope ( AFM) and fluorescence microscope ( FM) have been emerging as two most commonly used tools for single-molecule study in living cells. Combining the advantages of two microscopes, the development of the integrated AFM-FM technique with high spatiotemporal resolution and multi-function has attracted increasing interest. In this review, the principles of AFM single-molecular force spectroscopy and single-molecule fluorescence imaging were briefly discussed, and the recent advances in the integrated AFM-FM instrumentation were summarized. Subsequently based on our own research in the investigation of ligand-receptors interactions with the integrated AFM-FM technique, its applications in live-cell single-molecule imaging and characterization were introduced.

Engineered Protein Nanopore for Real-time Monitoring Single-molecule Reaction between Cadmium Ion and Glutathione / 分析化学

The chelating reaction between glutathione peptides and divalent cadmium ion was used as a typical model for investigating the coordination chemistry of SH-containing peptides and heavy metal ions, which was essential to understand the mechanism of intracellular cadmium detoxification.Here, the mutant (CM113R)7 αHL protein nanopore equipped with a first synthesized per-6-quaternary ammonium-β-cyclodextrin (p-QABCD) was used as nanoreactor and detector to investigate the single-molecule reaction of GSH molecules and Cd2+ ions.Different reaction pathways, intermediates, and products could be recognized.Cd2+-GSH reaction was highly dependent on the solution pH.The Cd(GSH)2 was formed at pH 7.4, while the Cd(GSH)2 and Cd2(GSH)2 were formed at pH 9.0.Cd2(GSH)2 was formed by two possible pathways: (1) A Cd2+ ion primarily coordinated with the thiol group of two GSH molecules to form Cd(GSH)2, and then the second Cd2+ ion quickly incorporated with the deprotonated amino group of Cd(GSH)2 to form Cd2(GSH)2;(2) Two Cd2+ ions separately coordinated with the thiol and deprotonated amino group of one GSH molecule to form Cd2(GSH)1, and the second GSH molecule quickly bounded to Cd2+ ions to form Cd2(GSH)2.This work studied the chelating reaction between metal ions and bioactive glutathione at single-molecule level without labeling and chemical modification, and would be important to further understand intracellular mechanisms of detoxification of heavy metals and greatly expand the research field of nanopore single-molecule technique.

Research Progresses in Single Molecule Enzymology / 分析化学

The single molecule imaging and technologies that developed in 1990 s have successfully probed the dynamics of single molecule enzyme catalysis in real time in vitro. Ever since then, single molecule enzymology has entered the golden age of rapid developing. Individual features of each enzyme hidden in the overall average have been discovered, and many new catalytic mechanisms have been proposed. Single molecule enzymology sheds light on the dynamic interactions between enzymes and substrates or products, deepening the understanding of biochemical reactions. This review described the recent research progresses of single molecule protease and ribozyme.

A Low Noise Amplifier System for Nanopore-based Single Molecule Analysis / 分析化学

A novel amplifier system was proposed for low-noise recording of pico-ampere current in nanopore experiment (<100 pA). As an example, the amplifier system was applied in α-hemolysin based nanopore detection of DNA-PEG-DNA conjugate to record the signals of translocation and bumping events in buffer solution (1 mol/L KCl, 10 mmol/L Tris--HCl, 1 mmol/L EDTA and pH=8. 0). The amplified current signal was filtered by a 3 kHz Bessel filter and sampled by a 100 kHz analog-digital convertor. As a result, the presented amplifier system could lower the noise in recording the current. The current blockages (<10 pA) of single molecules with low amplitude were recovered due to the high signal-to-noise ratio.

Effect of cigarette smoke extract on the interaction between thrombomodulin and thrombin by live-cell single-molecule force spectroscopy / 中国药理学通报

Aim To study the effect of CSE ( cigarette smoke extract ) on the single-molecule interactional force between thrombomodulin and thrombin by live-cell single-molecule force spectroscopy. Methods CSE was prepared by a previously reported method. The plasmid of TM-GFP was constructed and transfect-ed in COS-7 cells. The expression of TM-GFP was de-tected by fluorescence microscopy and laser scanning confocal microscopy. The transfected COS-7 cells were grouped ( 1 ) GFP -thrombin group ( 2 ) TM-thrombin group ( 3 ) CSE-TM-thrombin group ( 4 ) CSE- GFP-thrombin group. Force measurements with the thrombin modified AFM tips on the living cell surface were car-ried out on PicoSPM II with a Pico-Scan 3000 control-ler and a larger scanner. The force curves measured in living cells were recorded by PicoScan 5 software and analyzed by MATLAB R2009aMetlab. Results The single-molecule binding force of thrombomodulin and thrombin ( TM-Thr ) was determined ( 60. 90 ± 0. 82 ) pN. The binding probability for TM-Thr was about (22. 58 ± 3. 95)%. Antibody blocking binding proba-bility for TM-Thr was ( 2. 58 ± 2. 0 )%. The binding probabilities for GFP-Thr group, CSE-TM-Thr group and CSE-GFP-Thr group were significantly decreased compared with TM-Thr group ( P<0. 05 ) . The mean value of the most probable single molecular interaction force of thrombin/TM-ECD was determined as ( 45. 30 ± 1. 37 ) pN, the binding probability of thrombin and TM-ECD was ( 23. 25 ± 7. 02 )%. When the binding was blocked with the TM-MAb solution, the binding probability decreased to ( 4. 64 ± 2. 31 )%. The bind-ing probability was ( 8. 31 ± 1. 06 )% in the CSE-TM-thr-S group. When further blocked with TM-MAb, the binding probability was ( 5. 17 ± 2. 96 )%. Conclusion CSE significantly decreases the binding probability for TM-Thr to induce intravascular thrombosis.

Nanopore DNA single molecule sensing method and synthetic nanopore fabrication / 国际生物医学工程杂志

Nanopore single molecule sequencing(SMS) is considered as one of the most promising third generation DNA sequencing method by virtue of fast sequencing and low cost. As the most significant part of the sequencing system, the synthetic solid nanopore has recently been the research highlight in the field of nanopore DNA sequencing because of its fine-controlled size, high reliability and wide applicability. Also, its fabrication method is currently one of the main challenges in this field. In this article, the principles of the nanopore SMS are introduced followed by the review of the difficulties and challenges in the current research stage with emphasis on the nanopore fabrication methods.

Research progress in visualizing single molecule in live cells / 国际生物医学工程杂志

Our knowledge of molecular events and interactions in cell biology and molecular biology are mostly based on findings in nonliving body condition while ignoring the whole process of the events,which inevitably involves false assumption that all molecules behave in the same environment and in the same way. With the emergence of new fluorescent molecular probes prepared through linking new fluorescent material such as quantum dots (QDs) to micromolecule antibody such as antibody simulacrum, the development of methods for transducing extrinsic fluorescent probes into live cells,the application of laser technology, the finer fluorescent micro-imaging system, and the fast and sensitive fluorescence collection system in the application of life sciences, it is now possible to image the trajectory, kinetic behavior, and interaction of biomolecules in live cells. Visualizing single molecule in live cells has opened a new chapter for cell biology and molecular biology. Recent research developments and foreground of single molecule visualizing are discussed in this article.