Chunyi Tong

An Annexin V-based biosensor for quantitatively detecting early apoptotic cells

In this paper, we reported that a novel biosensor was developed to detect early apoptotic cells by the specific interaction between Annexin V and phosphatidylserine based on electrochemical impedance. Annexin V was immobilized on a self-assembled layer of gold nanoparticles, which allowed stable and high loading of Annexin V on the electrode surface, offering the possibility of sensitivity enhancement. Early apoptotic cells showed an increased exposition of phosphatidylserine on the cell membrane caused by physiological and pathological response reaction, leading to a strong interaction between the apoptotic cells and the electrode surface, which could be probed by electrochemical impedance spectroscopy. As examined using a model system of cells integrated by phosphatidylserine-modified liposome and a real one of early apoptotic cell induced by 5-fluorouracil, this biosensor demonstrated the great potential for rapid detection of cell apoptosis and drug screening. The results agreed well with those obtained using fluorescence microscopy and flow cytometry.

An end-point method based on graphene oxide for RNase H analysis and inhibitors screening

As a highly conserved damage repair protein, RNase H can hydrolysis DNA-RNA heteroduplex endonucleolytically and cleave RNA-DNA junctions as well. In this study, we have developed an accurate and sensitive RNase H assay based on fluorophore-labeled chimeric substrate hydrolysis and the differential affinity of graphene oxide on RNA strand with different length. This end-point measurement method can detect RNase H in a range of 0.01 to 1 units /mL with a detection limit of 5.0×10-3 units/ mL under optimal conditions. We demonstrate the utility of the assay by screening antibiotics, resulting in the identification of gentamycin, streptomycin and kanamycin as inhibitors with IC50 of 60±5µM, 70±8µM and 300±20µM, respectively. Furthermore, the assay was reliably used to detect RNase H in complicated biosamples and found that RNase H activity in tumor cells was inhibited by gentamycin and streptomycin sulfate in a concentration-dependent manner. The average level of RNase H in serums of HBV infection group was similar to that of control group. In summary, the assay provides an alternative tool for biochemical analysis for this enzyme and indicates the feasibility of high throughput screening inhibitors of RNase H in vitro and in vivo.

Preparation and functional characterization of tumor-targeted folic acid-chitosan conjugate nanoparticles loaded with mitoxantrone.

Folic acid conjugated chitosan was prepared by cross-linking reaction with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), and then used as a template to prepare folic acid-chitosan (FA–CS) conjugated nanoparticles and load mitoxantrone nanoparticles (FA-CSNP/MTX). Drug dissolution testing, CCK-8 method, and confocal microscopy were used to detect their controlled-release capability in different situations and the specific uptake by HONE1 cells. The experimental results show that the nanoparticles have uniform size distribution of 48–58 nm. The highest encapsulation rate of the particles on mitoxantrone hydrochloride (MTX) is (77.5±1.9)%, and the drug loading efficiency is (18.4±0.4)%. The sustained release effect, cell growth inhibition activity and targeting effect of the FA-CS/MTX nanoparticles are good in artificial gastric fluid and intestinal fluid. It is demonstrated that the FA-CSNP system is a potentially useful system for the targeted delivery of anticancer drug MTX.

Electrospun vein grafts with high cell infiltration for vascular tissue engineering

Demand is increasing for functional small-diameter vascular grafts (diameter<6mm) for clinical arterial replacement. In the present study, we develop a bilayer poly(ε-caprolactone, PCL) fibrous vascular graft consisting of a thin internal layer made of longitudinally aligned fibers and a relatively thick highly porous external layer. The internal layer provides a scaffold with the necessary mechanical strength and enhances the growth of endothelial cells, whereas the external layer enhances cell motility through the scaffold bulk. The biocompatibility and biological performance of bilayer fibrous scaffolds are evaluated by in vivo experiments, molecular biology, and histology studies. Our bilayer scaffolds demonstrate much better fiber alignment and higher porosity than do normal electrospun vascular grafts with randomly distributed fibers. The results suggest that the proposed grafts can overcome limitations owing to the inadequate porosity, small pores, and poor cell infiltration of scaffolds fabricated by conventional electrospinning. The unique structure of bilayer scaffolds is satisfactory and promotes cell proliferation, collagen-fiber deposition, and ingrowth of smooth muscle cells and endothelial cells in vivo. The results of this study illustrate the strong potential of such bilayer fibrous scaffolds for vascular tissue engineering and regeneration.

Isolation and evaluation of endophytic Streptomyces endus OsiSh-2 with potential application for biocontrol of rice blast disease

BACKGROUND Biocontrol is a promising strategy in the control of rice blast disease. In the present study, we isolated and characterized a novel antagonist to the pathogen Magnaporthe oryzae from rice endophytic actinomycetes. RESULTS Out of 482 endophytic actinomycetes isolated from rice blast infected and healthy rice, Streptomyces endus OsiSh-2 exhibited remarkable in vitro antagonistic activity. Scanning electron microscopy observations of M. oryzae treated by OsiSh-2 revealed significant morphological alterations in hyphae. In 2-year field tests, the spraying of OsiSh-2 spore solution (107  spores mL-1 ) is capable of reducing rice blast disease severity by 59.64%. In addition, a fermentation broth of OsiSh-2 and its cell-free filtrates could inhibit the growth of M. oryzae, suggesting the presence of active enzymes and secondary metabolites. OsiSh-2 tested positive for polyketide synthase-I and nonribosomal peptide synthetase genes and can produce cellulase, protease, gelatinase, siderophore, indole-3-acetic acid and 1-amino-cyclopropane-1-carboxylate deaminase. A preliminary separation indicated that the methanol extract of OsiSh-2 could suppress the growth of pathogens. The major active component was identified as nigericin. CONCLUSION Endophytic S. endus OsiSh-2 has potential as a biocontrol agent against rice blast in agriculture.

Sensitive Detection of RNase A Activity and Collaborative Drug Screening Based on rGO and Fluorescence Probe

In addition to being an important object in theoretical and experimental studies in enzymology, RNase A also plays an important role in the development of many kinds of diseases by regulating various physiological or pathological processes, including cell growth, proliferation, differentiation, and invasion. Thus, it can be used as a useful biomarker for disease theranostics. Here, a simple, sensitive, and low-cost assay for RNase A was constructed by combining a fluorogenic substrate with reduced graphene oxide (rGO). The method with detection limit of 0.05 ng/mL was first applied for RNase A targeted drug screening, and 14 natural compounds were identified as activators of this enzyme. Then, it was applied to detect the effect of drug treatment and Hepatitis B virus (HBV) infection on RNase A activity. The results indicated that RNase A level in tumor cells was upregulated by G-10 and Chikusetsusaponin V in a concentration-dependent manner, while the average level of RNase A in the HBV infection group was significantly inhibited compared with that in the control group. Furthermore, the concentration-dependent inhibitory effect of heavy metal ions on RNase A was observed using the method and the results indicated that Ba2+, Co2+, Pb2+, As3+, and Cu2+ inhibited RNase A activity with IC50 values of 93.7 μM (Ba2+), 90.9 μM (Co2+), 110.6 μM (Pb2+), 171.5 μM (As3+), and 165.1 μM (Cu2+), respectively. In summary, considering the benefits of rapidity and high sensitivity, the method is practicable for RNase A assay in biosamples and natural compounds screening in vitro and in vivo.

Increasing the sensitivity and selectivity of a GONS quenched probe for an mRNA assay assisted with duplex specific nuclease

The authors report a new graphene oxide nanosheet (GONS) based fluorescence method for mRNA assay with duplex-specific nuclease (DSN)-assisted signal amplification. Following hybridization between the target mRNA and its complementary DNA probe, the ssDNA in the DNA/mRNA hybrid is selectively cleaved by DSN to produce small fragments. The released mRNA molecule then initiates another cycle of hybridization and DSN digestion. In this manner, each mRNA molecule can specifically trigger many cycles of hybridization and DNA cleavages to produce numerous small DNA fragments. The short DNA fragments can exhibit strong fluorescence signals due to the weak adsorption of GONS to them. This fluorescence assay for mRNA with increased selectivity has a 1 fM detection limit under optimal conditions. Furthermore, this assay was successfully used for mRNA imaging in situ.

DNase-targeted natural product screening based on a sensitive and selective DNase I detecting system

As a widely used deoxyribonuclease, DNase I is involved in many physiological processes including tumor cell proliferation, metastasis and apoptosis. Furthermore, the level of this enzyme in serum can act as a functional biomarker for the therapeutic monitoring of systemic lupus erythematosus and other diseases. We report here a low cost and sensitive DNase I detecting system based on the single-stranded fluorogenic substrate and nanographene oxide (NGO) and use it for DNase-targeted natural product screening. The system with a detection limit of 0.005 U was then used to evaluate the effect of external factors on DNase I. The results show that Hg2+, As2+, Pb2+, Cd2+ and Cu2+ can inhibit DNase I activity in a concentration-dependent manner with IC50 values of 0.37 mM (Hg2+), 2.7 mM (As2+), 5 mM (Pb2+), 5.3 mM (Cd2+) and 7.8 mM (Cu2+), respectively. Meanwhile, 10 natural compounds isolated from Cyclocarya paliurus leaves were screened as DNase I inhibitors, while 5 compounds were identified as activators. Finally, the system was used to discriminate DNase activity of serum samples with and without HBV. The results showed that HBV infection significantly decreased the level of DNase I in serum samples. In summary, these data indicate that this method with the advantages of rapidity, low cost and high sensitivity is hopeful for DNase assay in biological samples as well as compound screening in vitro.

Synthesis of DNA-guided silver nanoparticles on a graphene oxide surface: enhancing the antibacterial effect and the wound healing activity

The occurrence of antibiotic resistance against pathogens is rapidly increasing and endangering the efficacy of antibiotics. Thus, finding a way to address this problem has become a major challenge due to the inability of conventional antibiotics to kill these multidrug-resistant bacteria. In order to further enhance the antibacterial ability and reduce the possibility of antibiotic resistance, we developed a simple two-step approach and synthesized a new nanocomposite by directly loading single-stranded DNA (ssDNA)-guided silver nanoparticles (AgNPs) on graphene oxide (ssDNA-AgNPs@GO). Through systematically evaluating the bactericidal activity and wound healing capability, we found that ssDNA-AgNPs@GO exhibited synergistic antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis with low minimum inhibitory concentrations (6.8 μg mL−1, 6.8 μg mL−1, 11.9 μg mL−1 and 10.2 μg mL−1, respectively) and large-diameter inhibition zones (12.83 ± 0.63 mm, 13.14 ± 0.37 mm, 8.6 ± 0.9 mm and 8.93 ± 0.47 mm, respectively). Furthermore, the wound healing experiment indicated that it has a striking ability to remedy wound infection caused by Staphylococcus aureus bacteria. In conclusion, the properties of ssDNA-AgNPs@GO with enhanced antibacterial and wound healing capability will give it broad applications in the future.

The Role of Iron Competition in the Antagonistic Action of the Rice Endophyte Streptomyces sporocinereus OsiSh-2 Against the Pathogen Magnaporthe oryzae

Rice blast caused by Magnaporthe oryzae severely impacts global rice yield stability. The rice endophyte Streptomyces sporocinereus OsiSh-2, with strong antagonistic activity towards M. oryzae, has been reported in our previous study. To decipher the model of the antagonistic action of OsiSh-2 towards M. oryzae, we compared the iron-capturing abilities of these two strains. The cultivation of OsiSh-2 and a M. oryzae strain under iron-rich and iron-starved conditions showed that M. oryzae depended more on iron supplementation for growth and development than did OsiSh-2. Genomic analysis of the S. sporocinereus and M. oryzae species strains revealed that they might possess different iron acquisition strategies. The actinobacterium OsiSh-2 is likely to favor siderophore utilization compared to the fungus M. oryzae. In addition, protein annotations found that OsiSh-2 contains the highest number of the siderophore biosynthetic gene clusters among the 13 endophytic actinomycete strains and 13 antifungal actinomycete strains that we compared, indicating the prominent siderophore production potential of OsiSh-2. Additionally, we verified that OsiSh-2 could excrete considerably more siderophores than Guy11 under iron-restricted conditions and displayed greater Fe3+-reducing activity during iron-supplemental conditions. Measurements of the iron mobilization between the antagonistic OsiSh-2 and Guy11 showed that the iron concentration is higher around OsiSh-2 than around Guy11. In addition, adding iron near OsiSh-2 could decrease the antagonism of OsiSh-2 towards Guy11. Our study revealed that the antagonistic capacity displayed by OsiSh-2 towards M. oryzae was related to the competition for iron. The highly efficient iron acquisition system of OsiSh-2 may offer valuable insight for the biocontrol of rice blast.