Fast LC-MS quantification of ptesculentoside, caudatoside, ptaquiloside and corresponding pterosins in bracken ferns.

Ptaquiloside (PTA) is an illudane glycoside partly responsible for the carcinogenicity of bracken ferns (Pteridium sp.). The PTA analogues ptesculentoside (PTE) and caudatoside (CAU) have similar biochemical reactivity. However, both compounds are highly under-investigated due to the lack of analytical standards and appropriate methods. This study presents a robust method for preparation of analytical standards of PTE, CAU, PTA, the corresponding hydrolysis products: pterosins G, A and B, and an LC-MS based method for simultaneous quantification of the six compounds in bracken. The chromatographic separation of analytes takes 5 min. The observed linear range of quantification was 20-500 µg/L for PTA and pterosin B, and 10-250 µg/L for the remaining compounds (r > 0.999). The limits of detection were 0.08-0.26 µg/L for PTE, CAU and PTA and 0.01-0.03 µg/L for the pterosins, equivalent to 2.0-6.5 µg/g and 0.25-0.75 µg/g in dry weight, respectively. The method was applied on 18 samples of dried fern leaves from 6 continents. Results demonstrated high variation in concentrations of PTE, CAU and PTA with levels prior to hydrolysis up to 3,900, 2,200 and 2,100 µg/g respectively. This is the first analytical method for simultaneous and direct measurement of all six compounds. Its application demonstrated that bracken ferns contain significant amounts of PTE and CAU relative to PTA.

Ultrasmall TPGS-PLGA Hybrid Nanoparticles for Site-Specific Delivery of Antibiotics into Pseudomonas aeruginosa Biofilms in Lungs.

Inhaled antibiotic treatment of cystic fibrosis-related bacterial biofilm infections is challenging because of the pathological environment of the lungs. Here, we present an "environment-adaptive" nanoparticle composed of a solid poly lactic-co-glycolic acid (PLGA) core and a mucus-inert, enzymatically cleavable shell of d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) for the site-specific delivery of antibiotics to bacterial biofilms via aerosol administration. The hybrid nanoparticles with ultrasmall size were self-assembled via a nanoprecipitation process by using a facile microfluidic method. The interactions of the nanoparticles with the biological barriers were comprehensively investigated by using cutting-edge techniques (e.g., quartz crystal microbalance with dissipation monitoring, total internal reflection fluorescence microscopy-based particle tracking, in vitro biofilm model cultured in a flow-chamber system, and quantitative imaging analysis). Our results suggest that the mucus-inert, enzymatically cleavable TPGS shell enables the nanoparticles to penetrate through the mucus, accumulate in the deeper layer of the biofilms, and serve as sustained release depot, thereby improving the killing efficacy of azithromycin (a macrolide antibiotic) against biofilm-forming Pseudomonas aeruginosa. In conclusion, the ultrasmall TPGS-PLGA hybrid nanoparticles represent an efficient delivery system to overcome the multiple barriers and release antibiotics in a sustained manner in the vicinity of the biofilm-forming bacteria.

Epidermal LysM receptor ensures robust symbiotic signalling in Lotus japonicus.

Recognition of Nod factors by LysM receptors is crucial for nitrogen-fixing symbiosis in most legumes. The large families of LysM receptors in legumes suggest concerted functions, yet only NFR1 and NFR5 and their closest homologs are known to be required. Here we show that an epidermal LysM receptor (NFRe), ensures robust signalling in L. japonicus. Mutants of Nfre react to Nod factors with increased calcium spiking interval, reduced transcriptional response and fewer nodules in the presence of rhizobia. NFRe has an active kinase capable of phosphorylating NFR5, which in turn, controls NFRe downstream signalling. Our findings provide evidence for a more complex Nod factor signalling mechanism than previously anticipated. The spatio-temporal interplay between Nfre and Nfr1, and their divergent signalling through distinct kinases suggests the presence of an NFRe-mediated idling state keeping the epidermal cells of the expanding root system attuned to rhizobia.