In vivo femtosecond laser nanosurgery of the cell wall enabling patch-clamp measurements on filamentous fungi

Studying the membrane physiology of filamentous fungi is key to understanding their interactions with the environment and crucial for developing new therapeutic strategies for disease-causing pathogens.However,their plasma membrane has been inaccessible for a micron-sized patch-clamp pipette for pA current recordings due to the rigid chitinous cell wall.Here,we report the first femtosecond IR laser nanosurgery of the cell wall of the filamentous fungi,which enabled patch-clamp measurements on protoplasts released from hyphae.A reproducible and highly precise(diffraction-limited,submicron resolution)method for obtaining viable released protoplasts was developed.Protoplast release from the nanosurgery-generated incisions in the cell wall was achieved from different regions of the hyphae.The plasma membrane of the obtained protoplasts formed tight and high-resistance(GΩ)contacts with the recording pipette.The entire nanosurgical procedure followed by the patch-clamp technique could be completed in less than 1 hour.Compared to previous studies using heterologously expressed channels,this technique provides the opportunity to identify new ionic currents and to study the properties of the ion channels in the protoplasts of filamentous fungi in their native environment.

Sharing roosts but not ectoparasites:high host-specificity in bat flies and wing mites of Miniopterus schreibersii and Rhinolophus ferrumequinum(Mammalia:Chiroptera)

Schreiber’s bent-winged bat Miniopterus schreibersii and the greater horseshoe bat Rhinolophus ferrumequinum are widespread and common cavernicolous species across southern Europe that host numerous specialized ectoparasite species.The objective of this study was to characterize the species assemblage,genetic diversity,and host specificity of bat flies(Nycteribiidae,Diptera)and wing mites(Spinturnicidae,Acari)found on these bat hosts in Serbia and Bosnia and Herzegovina.Notably,while bat flies lay puparia on the cave walls and can thus be transmitted indirectly,wing mites require direct body contact for transmission.Morphological identification and sequencing of a 710-bp fragment of cytochrome oxidase I gene of 207 bat flies yielded 4 species,3 on M.schreibersii and 1 on R.ferrumequinum.Sequencing of a 460-bp small subunit ribosomal RNA fragment,in all 190 collected wing mites revealed 2 species,1 per host.In no case was a parasite associated with 1 host found on the other host.Species and genetic diversity of flies were higher in M.schreibersii,likely reflecting their host’s larger colony sizes and migratory potential.Mite species of both hosts showed similarly low diversity,likely due to their faster life history and lower winter survival.Our findings highlight a remarkably high host-specificity and segregation of ectoparasite species despite direct contact among their hosts in the roost,suggesting a defined host preference in the investigated ectoparasite species.Furthermore,the differences in ectoparasite genetic diversity exemplify the interplay between host and parasite life histories in shaping parasite population genetic structure.

Shining the spotlight on NMR metabolic profiling and bioactivities of different solvent extracts of Piliostigma thonningii

Piliostigma thonningii (Fabaceae) is a versatile medicinal plant used as a traditional remedy,especially in African countries.In the present study,ethyl acetate,methanolic and water extracts of different parts of (fruits,leaves,stem barks) P.thonningii were evaluated for their bioactive contents,enzyme inhibitory,antioxidant and antimicrobial activities.Antioxidant potentials were also displayed in various methods like phosphomolybdenum assay (0.79–5.94 mmol TE/g),radical scavenging assays (DPPH:20.39–471.28 mg TE/g;ABTS:42.43–654.14 mg TE/g);reducing assays (CUPRAC:102.98–1207.42 mg TE/g;FRAP:45.30–656.25 mg TE/g) and metal chelating activity (2.55–22.32 mg EDTAE/g).In particular,the methanolic and water extracts showed better antioxidant potential.Except for a few,the extracts were also found to act as inhibitors of enzymes such as acetylcholinesterase (2.35–5.35 mg GALAE/g),butyrylcholinesterase (0.83–13.80 mg GALAE/g),tyrosinase (4.03–143.86 mg KAE/g),amylase (0.21–1.39 mmol ACAE/g) and glucosidase (3.52–7.13 mmol ACAE/g).Additionally,all the tested extracts possessed significant antibacterial (minimum inhibitory concentration (MIC) and minimum bactericidal concentration:0.11–0.61 mg/ml) and antifungal (MIC and minimum fungicidal concentration:0.04–0.61 mg/ml) effects.Indeed,the data garnered from the present study provides a bright scope for the advanced investigation on this particular plant possessing pharmacologically active traits.

Phytochemical characterisation, in vitro antioxidant and antidiabetic activity of Rosa arvensis Huds. extracts

In this study, the chemical composition, antioxidant, and hypoglycemic activity of Rosa arvensis Huds. leaves and fruits collected from Zlatibor, Željin, Veliki Krš mountains and Petnica village (Serbia) were analysed. Phenolic and vitamin profiles, as well as in vitro antioxidant potential and enzyme inhibitory activity on α-amylase and α-glucosidase, were studied. Phytochemical analysis showed higher phenolic content in leaf extracts than in those obtained from fruits. Gallic, chlorogenic and syringic acids were the most abundant phenolic acids, while catechin was the major flavonoid compound. Furthermore, the highest level of α-tocopherol (1.94 mg/100 g fw) in achenes and ascorbic acid (9.60 mg/100 g fw) in hypanthium were quantified in the fruits sampled on the Veliki Krš Mt. High antioxidant capacity was observed for the leaf extracts. It was highly correlated with their chemical composition and primarily referred to the catechin content (r > 0.95, p < 0.01), while the quinic acid had the opposite effect (r > −0.97, p < 0.01). Leaf extracts also showed good α-glucosidase and moderate α-amylase inhibitory potential in contrast to hypanthium and achenes extracts with lower antidiabetic activity. The high phenolic content and noted antioxidative potential were strongly correlated with the α-glucosidase inhibitory activity (r > 0.80, p < 0.01), while the α-amylase inhibition was only moderately correlated with those compounds (r > 0.49, p < 0.01). Among the tested R. arvensis samples, those from Zlatibor Mt. contained the highest amount of target compounds and expressed the highest biological activity. The obtained results suggest that R. arvensis extracts present a new source of bioactive compounds that could be implemented as novel food ingredients.