Xue Peng

Synthesis and in vitro antifungal efficacy of oleoyl-chitosan nanoparticles against plant pathogenic fungi

An antifungal dispersion system was prepared by oleoyl-chitosan (O-chitosan) nanoparticles, and the antifungal activity against several plant pathogenic fungi was investigated. Under scanning electron microscopy, the nanoparticles formulation appeared to be uniform with almost spherical shape. The particle size of nanoparticles was around 296.962 nm. Transmission electron microscopy observation showed that nanoparticles could be well distributed in potato dextrose agar medium. Mycelium growth experiment demonstrated that Nigrospora sphaerica, Botryosphaeria dothidea, Nigrospora oryzae and Alternaria tenuissima were chitosan-sensitive, while Gibberella zeae and Fusarium culmorum were chitosan-resistant. The antifungal index was increased as the concentration of nanoparticles increased for chitosan-sensitive fungi. Fatty acid analyses revealed that plasma membranes of chitosan-sensitive fungi were shown to have lower levels of unsaturated fatty acid than chitosan-resistant fungi. Phylogenetic analysis based on ITS gene sequences indicated that two chitosan-resistant fungi had a near phylogenetic relationship. Results showed that O-chitosan nanoparticles could be a useful alternative for controlling pathogenic fungi in agriculture.

Ultrasensitive electrochemical sensor for Hg(2+) by using hybridization chain reaction coupled with Ag@Au core-shell nanoparticles.

A novel electrochemical biosensor for Hg(2+) detection was reported by using DNA-based hybridization chain reaction (HCR) coupled with positively charged Ag@Au core-shell nanoparticles ((+)Ag@Au CSNPs) amplification. To construct the sensor, capture probe (CP ) was firstly immobilized onto the surface of glass carbon electrode (GCE). In the presence of Hg(2+), the sandwiched complex can be formed between the immobilized CP on the electrode surface and the detection probe (DP) modified on the gold nanoparticles (AuNPs) based on T-Hg(2+)-T coordination chemistry. The carried DP then opened two ferrocene (Fc) modified hairpin DNA (H1 and H2) in sequence and propagated the happen of HCR to form a nicked double-helix. Numerous Fc molecules were formed on the neighboring probe and produced an obvious electrochemical signal. Moreover, (+)Ag@Au CSNPs were assembly onto such dsDNA polymers as electrochemical signal enhancer. Under optimal conditions, such sensor presents good electrochemical responses for Hg(2+) detection with a detection limit of 3.6 pM. Importantly, the methodology has high selectivity for Hg(2+) detection.

Ultrasensitive electrochemical detection of protein tyrosine kinase-7 by gold nanoparticles and methylene blue assisted signal amplification

We present here an ultrasensitive and simple strategy for protein tyrosine kinase-7 (PTK7) detection based on the recognition-induced structure change of sgc8 aptamer, and the signal change of methylene blue (MB) that interacted with sandwiched DNA complex. To construct such a sensor, an homogeneous nano-surface was formed firstly on the glass carbon electrode (GCE) by using negatively charged Nafion (Nf) as the inner layer and positively charged gold nanoparticles ((+)AuNPs) as the outer layer, followed by the immobilization of sgc8 aptamer based on Au-S bond. In the presence of helper probe (HP), sandwiched DNA complex was formed between the sgc8 aptamer and the DNA modified gold nanoparticle probe (DNA-AuNPs). Then, a strong current signal was produced due to the capture of abundant MB molecules by both the sandwiched DNA complex and the multiple DNAs that modified on AuNPs surface. However, the specific binding of sgc8 aptamer with PNK7 would trigger a structure transition of it, and directly prevented the following formation of sandwiched structure and the capture of MB. Thus, PTK7 detection could be realized based on monitoring the signal reduction of MB upon incubation of sgc8 aptamer with PTK7. Under optimal conditions, a low detection limit of 372 fM was obtained for PNK7 detection. Due to the employment of sgc8 aptamer, the proposed biosensor exhibited high selectivity to PNK7. Moreover, satisfactory results were obtained when the proposed method was applied for PNK7 detection in cellular debris.

Efficacy and possible mechanisms of perillaldehyde in control of Aspergillus niger causing grape decay

A variety of plant products have been recognized for their antifungal activity and recently have attracted food industry attention for their efficacy in controlling postharvest fungal decay of fruits. The antifungal activity of perillaldehyde (PAE) was evaluated against Aspergillus niger, a known cause of grape spoilage, and possible mechanisms were explored. PAE showed notable antifungal activity against A. niger, with a minimum inhibitory concentration (MIC) and a minimum fungicidal concentration (MFC) of 0.25 and 1 μl/ml, respectively. The accumulation of mycelial biomass was also inhibited by PAE in a dose-dependent manner, completely inhibiting mycelial growth at 1 μl/ml. In vivo data confirmed that the vapour treatment of grapes with various concentrations of PAE markedly improved control of A. niger and suppressed natural decay. Concentrations of PAE of 0.075 μl/ml air showed the greatest inhibition of fungal growth compared to the controls. Further experiments indicated that PAE activated a membrane-active mechanism that inhibits ergosterol synthesis, increases membrane permeability (as evidenced by extracellular pH and conductivity measurements), and disrupts membrane integrity, leading to cell death. Our findings suggest that this membrane-active mechanism makes PAE a promising potential antifungal agent for postharvest control of grape spoilage.

Nerol triggers mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS in Candida albicans

The antifungal activity of Nerol (NEL) against Candida albicans, a pathogenic fungus, has a minimum inhibitory concentration (MIC) of 4.4mM that causes noteworthy candidacidal activity through an apoptosis-like mechanism. Calcium (Ca2+) levels and reactive oxygen species (ROS) production, which are the major causes of apoptosis, were determined in C. albicans cells treated with NEL and were found to increase, which related to mitochondrial dysfunction and disruption. A series of characteristic changes of apoptosis caused by NEL, including mitochondrial membrane depolarization, cytochrome c (cyt c) release, and metacaspase activation were examined using a flow cytometer and Western blot. The results showed that an increase in intracellular Ca2+ and ROS led to dramatically decreased mitochondrial membrane potential (MMP); cyt c was also released from the mitochondria to the cytosol. Other early apoptotic features were also observed with the metacaspase activation. Finally, the morphological changes of the cells were observed, including phosphatidylserine (PS) externalization, nuclear condensation, and DNA fragmentation through Annexin V-FITC and PI double staining, TUNEL assay, and DAPI staining. The results supported the hypothesis that NEL was involved in the apoptosis of C. albicans cells not only at the early stages, but also at the late stages. In summary, NEL can trigger mitochondrial dysfunction and disruption via elevation of Ca2+ and ROS leading to apoptosis in C. albicans. This research on the mechanism of cell death triggered by NEL against C. albicans has important significance for providing a novel treatment of C. albicans infections.

Investigations on the Antifungal Effect of Nerol against Aspergillus flavus Causing Food Spoilage

The antifungal efficacy of nerol (NEL) has been proved against Aspergillus flavus by using in vitro and in vivo tests. The mycelial growth of A. flavus was completely inhibited at concentrations of 0.8u2009μL/mL and 0.1u2009μL/mL NEL in the air at contact and vapor conditions, respectively. The NEL also had an evident inhibitory effect on spore germination in A. flavus along with NEL concentration as well as time-dependent kinetic inhibition. The NEL presented noticeable inhibition on dry mycelium weight and synthesis of aflatoxin B1 (AFB1) by A. flavus, totally restraining AFB1 production at 0.6u2009μL/mL. In real food system, the efficacy of the NEL on resistance to decay development in cherry tomatoes was investigated in vivo by exposing inoculated and control fruit groups to NEL vapor at different concentration. NEL vapors at 0.1u2009μL/mL air concentration significantly reduced artificially contaminated A. flavus and a broad spectrum of fungal microbiota. Results obtained from presented study showed that the NEL had a great antifungal activity and could be considered as a benefit and safe tool to control food spoilage.

Effect of O-chitosan nanoparticles on the development and membrane permeability of Verticillium dahliae

Verticillium dahliae, which causes wilting in over 300 woody and herbaceous plant species, is a representative of fungal plant diseases for which effective controls are still needed. In this study, the antifungal action of oleoyl-chitosan nanoparticles was investigated against V. dahliae. Media containing oleoyl-chitosan nanoparticles dramatically decreased the mycelium growth. The highest antifungal indexes were observed on media amended with 2mg/mL nanoparticles. Optical microscopy showed that spore germination and hyphae morphology were affected. Scanning electron microscopy and transmission electron microscopy demonstrated degenerative alterations including crumpled hyphae and spores, thickened cell walls, disappeared membranous organelles, massive vacuolation of the cytoplasm, and cell wall-plasmalemma separation. Fluorescence microscopy showed that nanoparticles were internalized by fungal cells. The sharp increase in the release of intracellular components and decrease of total cellular protein concentration demonstrated damaged cell membranes. Overall, the results indicate that oleoyl-chitosan nanoparticles have the potential to control phytopathogens in agriculture.

Glucose detection at attomole levels using dynamic light scattering and gold nanoparticles

Glucose is directly related to brain activity and to diabetes. Therefore, developing a rapid and sensitive method for glucose detection is essential. Here, label-free glucose detection at attomole levels was realized by detecting the average diameter change of gold nanoparticles (AuNPs) utilizing dynamic light scattering (DLS). Single-strand DNA (ssDNA) adsorbed into the AuNPs’ surfaces and prevented them from aggregating in solution that contained NaCl. However, ssDNA cleaved onto ssDNA fragments upon addition of glucose, and these fragments could not adsorb onto the AuNPs’ surfaces. Therefore, in high-salt solution, AuNPs would aggregate and their average diameter would increase. Based on monitoring the average diameter of AuNPs with DLS, glucose could be detected in the range from 15 pmol/L to 2.0 nmol/L, with a detection limit of 8.3 pmol/L. Satisfactory results were also obtained when the proposed method was applied in human serum glucose detection.

Glutamicibacter halophytocola sp. nov., an endophytic actinomycete isolated from the roots of a coastal halophyte, Limonium sinense

A novel actinobacterium, designated KLBMP 5180T, was isolated from the surface-sterilized root of a coastal halophyte, Limonium sinense, collected from the city of Lianyungang, Jiangsu Province, eastern China. The isolate was Gram-stain-positive, aerobic and non-motile. The components of the cell-wall peptidoglycan were lysine, glutamic acid and alanine. The predominant menaquinone was MK-9. The polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylinositol, one unknown phospholipid, one unidentified glycolipid and two unidentified lipids. anteiso-C15u200a:u200a0 and iso-C16u200a:u200a0 were the major cellular fatty acids. The DNA G+C content of strain KLBMP 5180T was 60.0u2009mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain KLBMP 5180T belongs to the genus Glutamicibacter and was related most closely to Glutamicibacter nicotianae DSM 20123T (99.3u200a% similarity), Glutamicibacterarilaitensis Re117T (99.3u200a%) and Glutamicibacter mysorens LMG 16219T (99.1u200a%); similarity to other type strains of the genus Glutamicibacter was lower than 98.5u200a%. However, DNA-DNA relatedness values between strain KLBMP 5180T, G . nicotianae DSM 20123T, G. arilaitensis Re117T and G. mysorens LMG 16219T were 47.5±2.6, 51.3±3.1u2009and 41.2±4.3u200a%, respectively. The combination of DNA-DNA hybridization, phylogenetic, phenotypic and chemotaxonomic data supported the suggestion that strain KLBMP 5180T represents a novel species of the genus Glutamicibacter, for which the name Glutamicibacterhalophytocola sp. nov. is proposed. The type strain is KLBMP 5180T (=DSM 101718T=KCTC 39692T).

Fungicidal effect of chitosan via inducing membrane disturbance against Ceratocystis fimbriata

In this study, the fungicidal effects and detailed action of chitosan against Ceratocystis fimbriata were evaluated. The results demonstrated that chitosan exhibited strong antifungal activity that restricted the mycelium extension and changed the hyphal morphology. Fluorescein diacetate (FDA) and propidium iodide (PI) double-staining directly visualized decreased cell viability in response to chitosan treatment. Investigation of the PI influx showed that chitosan induced irreversible cell membrane damage. The efflux of potassium ions from the cytosol into the extracellular matrix demonstrated that chitosan induced the leakage of intracellular components. Massive intracellular bis-(1, 3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] accumulation indicated the dissipation of membrane potential. Furthermore, chitosan clearly decreased the activity of H+/K+ ATPase. Fluorescence microscopy revealed that the fluorescence distribution and intensity of fluorescein isothiocyanate (FITC) changed as the incubation time increased. These results indicate that chitosan exerts a fungicidal effect via its ability to disturb fungal membranes.