The quorum sensing (QS) inhibitory activity of hordenine from sprouting barley against foodborne pathogen Pseudomonas aeruginosa was evaluated for the first time here. At concentrations ranging from 0.5 to 1.0 mg mL, hordenine inhibited the levels of acyl-homoserine lactones. The enhanced susceptibility of hordenine with netilmicin on P. aeruginosa PAO1 biofilm formation as well as their efficiency in disrupting preformed biofilms was also evaluated using scanning electron microscopy and confocal laser scanning microscopy (CLSM). Hordenine treatment inhibited the production of QS-related extracellular virulence factors of P. aeruginosa PAO1. Additionally, quantitative real-time polymerase chain reaction analysis demonstrated that the expressions of QS-related genes, lasI, lasR, rhlI, and rhlR, were significantly suppressed. Our results indicated that hordenine can serve as a competitive inhibitor for signaling molecules and act as a novel QS-based agent to defend against foodborne pathogens.

No abstract

This study reported the efficacy of the metabolites of Plectosphaerella cucumerina, one phyllosphere fungus from Orychophragmus violaceus, against Pseudomonas aeruginosa quorum sensing (QS) and QS-regulated biofilms. The minimum inhibitory concentration (MIC) of the ethyl acetate (EtOAc) extract from P. cucumerina against P. aeruginosa PAO1 was 1.25 mg mL−1. At sub-MIC concentrations, P. cucumerina extract (0.25–1 mg mL−1) not only inhibited biofilm formation but also disrupted preformed biofilms of P. aeruginosa PAO1 without affecting its growth. Fluorescence and scanning electron microscope (SEM) showed architectural disruption of the biofilms when treated with P. cucumerina metabolites. Further investigation demonstrated that metabolites in P. cucumerina attenuated the QS-dependent virulence factors. LC-MS/MS spectra coupled with experimentally standard samples suggested that patulin and emodin might act as the principal components possessing anti-biofilm and antivirulence activities. This is the first report of (1) the isolation of P. cucumerina from the phyllosphere of O. violaceus and (2) anti-biofilm, antivirulence, and biofilm disruption activities of this fungus. Thus, this study provides fascinating new pathways for screening antipathogenic agents.

No abstract

No abstract

Genetic engineering in livestock was greatly enhanced by the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9), which can be programmed with a single-guide RNA (sgRNA) to generate site-specific DNA breaks. However, the uncertainties caused by wide variations in sgRNA activity impede the utility of this system in generating genetically modified pigs. Here, we described a single blastocyst genotyping system to provide a simple and rapid solution to evaluate and compare the sgRNA efficiency at inducing indel mutations for a given gene locus. Assessment of sgRNA mutagenesis efficiencies can be achieved within 10 days from the design of the sgRNA. The most effective sgRNA selected by this system was successfully used to induce site-specific insertion through homology-directed repair at a frequency exceeding 13%. Additionally, the highly efficient gene deletion via the selected sgRNA was confirmed in pig fibroblast cells, which could serve as donor cells for somatic cell nuclear transfer. We further showed that direct cytoplasmic injection of Cas9 mRNA and the favorable sgRNA into zygotes could generate biallelic knockout piglets with an efficiency of up to 100%. Thus, our method considerably reduces the uncertainties and expands the practical possibilities of CRISPR/Cas9-mediated genome engineering in pigs.

Transient photocurrent experiments are used to measure the free radical ion quantum yield of a number of alkylbenzene electron donors with the electron acceptor tetracyanoethylene (TCNE). These experiments are performed at a variety of photoexcitation wavelengths in dichloromethane, a moderately polar solvent. It is found that the free ion yields often exhibit a very strong dependence on the excitation wavelength and may decrease markedly in the center of the charge-transfer band. For example, the free ion yield of the donoracceptor system, pentamethylbenzene-TCNE, increases more than 100-fold when the excitation wavelength is switched from 532 to 397 nm! We show that this result and others are understandable from the following model. While closely associated electron donor-acceptor (EDA) complexes account for most of the absorption, there is an additional, usually small, absorption due to unassociated random donor and acceptor pairs. The Franck-Condon (vertical) excitation of these random pairs results in radical ion pairs which have centerto-center distances greater than contact and which have high probabilities for separation. Quantitative analysis based on Onsager theory indicates that only distantly separated radical ion pairs (ca. 1 nm or more) created by photoexcitation can escape each other's Coulombic attraction to produce the free ion yields observed in our experiments. The photoexcitation of ground-state EDA complexes plays little essential role in this process. The observed wavelength dependence then corresponds both to variation in the ratio of random pair to EDA complex absorption and to the distance distribution of radical ion pairs produced. Free ion yields calculated using Onsager theory and a simple excitation function for the random pairs fit our experimental results quite well and support this model.

No abstract