Strong antagonism of an endophyte of Fraxinus excelsior towards the ash dieback pathogen, Hymenoscyphus fraxineus, is mediated by the antifungal secondary metabolite PF1140.

Ash dieback, caused by the fungal pathogen Hymenoscyphus fraxineus (Helotiales, Ascomycota), is threatening the existence of the European ash, Fraxineus excelsior. During our search for biological control agents for this devastating disease, endophytic fungi were isolated from healthy plant tissues and co-cultivated with H. fraxineus to assess their antagonistic potential. Among the strains screened, Penicillium cf. manginii DSM 104493 most strongly inhibited the pathogen. Initially, DSM 104493 showed promise in planta as a biocontrol agent. Inoculation of DSM 104493 into axenically cultured ash seedlings greatly decreased the development of disease symptoms in seedlings infected with H. fraxineus. The fungus was thus cultivated on a larger scale in order to obtain sufficient material to identify active metabolites that accounted for the antibiosis observed in dual culture. We isolated PF1140 (1) and identified it as the main active compound in the course of a bioassay-guided isolation strategy. Furthermore, its derivative 2, the mycotoxin citreoviridin (3), three tetramic acids of the vancouverone type (4-6), and penidiamide (7) were isolated by preparative chromatography. The structures were elucidated mainly by NMR spectroscopy and high-resolution mass spectrometry (HRMS), of which compounds 2 and 6 represent novel natural products. Of the compounds tested, not only PF1140 (1) strongly inhibited H. fraxineus in an agar diffusion assay but also showed phytotoxic effects in a leaf puncture assay. Unfortunately, both the latent virulent attributes of DSM 104493 observed subsequent to these experiments in planta and the production of mycotoxins exclude strain Penicillium cf. manginii DSM 104493 from further development as a safe biocontrol agent.IMPORTANCEEnvironmentally friendly measures are urgently needed to control the causative agent of ash dieback, Hymenoscyphus fraxineus. Herein, we show that the endophyte DSM 104493 exhibits protective effects in vitro and in planta. We traced the activity of DSM 104493 to the antifungal natural product PF1140, which unfortunately also showed phytotoxic effects. Our results have important implications for understanding plant-fungal interactions mediated by secondary metabolites, not only in the context of ash dieback but also generally in plant-microbial interactions.

Structured illumination ptychography and at-wavelength characterization with an EUV diffuser at 13.5†nm wavelength.

Structured illumination is essential for high-performance ptychography. Especially in the extreme ultraviolet (EUV) range, where reflective optics are prevalent, the generation of structured beams is challenging and, so far, mostly amplitude-only masks have been used. In this study, we generate a highly structured beam using a phase-shifting diffuser optimized for 13.5 nm wavelength and apply this beam to EUV ptychography. This tailored illumination significantly enhances the quality and resolution of the ptychography reconstructions. In particular, when utilizing the full dynamics range of the detector, the resolution has been improved from 125 nm, when using an unstructured beam, to 34 nm. Further, ptychography enables the quantitative measurement of both the amplitude and phase of the EUV diffuser at 13.5 nm wavelength. This capability allows us to evaluate the influence of imperfections and contaminations on its "at wavelength" performance, paving the way for advanced EUV metrology applications and highlighting its importance for future developments in nanolithography and related fields.

Differential transcriptomic host responses in the early phase of viral and bacterial infections in human lung tissue explants ex vivo.

BACKGROUND: The first 24 h of infection represent a critical time window in interactions between pathogens and host tissue. However, it is not possible to study such early events in human lung during natural infection due to lack of clinical access to tissue this early in infection. We, therefore, applied RNA sequencing to ex vivo cultured human lung tissue explants (HLTE) from patients with emphysema to study global changes in small noncoding RNA, mRNA, and long noncoding RNA (lncRNA, lincRNA) populations during the first 24 h of infection with influenza A virus (IAV), Mycobacterium bovis Bacille Calmette-Guerin (BCG), and Pseudomonas aeruginosa. RESULTS: Pseudomonas aeruginosa caused the strongest expression changes and was the only pathogen that notably affected expression of microRNA and PIWI-associated RNA. The major classes of long RNAs (> 100 nt) were represented similarly among the RNAs that were differentially expressed upon infection with the three pathogens (mRNA 77-82%; lncRNA 15-17%; pseudogenes 4-5%), but lnc-DDX60-1, RP11-202G18.1, and lnc-THOC3-2 were part of an RNA signature (additionally containing SNX10 and SLC8A1) specifically associated with IAV infection. IAV infection induced brisk interferon responses, CCL8 being the most strongly upregulated mRNA. Single-cell RNA sequencing identified airway epithelial cells and macrophages as the predominant IAV host cells, but inflammatory responses were also detected in cell types expressing few or no IAV transcripts. Combined analysis of bulk and single-cell RNAseq data identified a set of 6 mRNAs (IFI6, IFI44L, IRF7, ISG15, MX1, MX2) as the core transcriptomic response to IAV infection. The two bacterial pathogens induced qualitatively very similar changes in mRNA expression and predicted signaling pathways, but the magnitude of change was greater in P. aeruginosa infection. Upregulation of GJB2, VNN1, DUSP4, SerpinB7, and IL10, and downregulation of PKMYT1, S100A4, GGTA1P, and SLC22A31 were most strongly associated with bacterial infection. CONCLUSIONS: Human lung tissue mounted substantially different transcriptomic responses to infection by IAV than by BCG and P. aeruginosa, whereas responses to these two divergent bacterial pathogens were surprisingly similar. This HLTE model should prove useful for RNA-directed pathogenesis research and tissue biomarker discovery during the early phase of infections, both at the tissue and single-cell level.

Honey bee colonies can buffer short-term stressor effects of pollen restriction and fungicide exposure on colony development and the microbiome.

Honey bees (Apis mellifera) have to withstand various environmental stressors alone or in combination in agriculture settings. Plant protection products are applied to achieve high crop yield, but residues of their active substances are frequently detected in bee matrices and could affect honey bee colonies. In addition, intensified agriculture could lead to resource limitation for honey bees. This study aimed to compare the response of full-sized and nucleus colonies to the combined stressors of fungicide exposure and resource limitation. A large-scale field study was conducted simultaneously at five different locations across Germany, starting in spring 2022 and continuing through spring 2023. The fungicide formulation Pictor® Active (active ingredients boscalid and pyraclostrobin) was applied according to label instructions at the maximum recommended rate on oil seed rape crops. Resource limitation was ensured by pollen restriction using a pollen trap and stressor responses were evaluated by assessing colony development, brood development, and core gut microbiome alterations. Furthermore, effects on the plant nectar microbiome were assessed since nectar inhabiting yeast are beneficial for pollination. We showed, that honey bee colonies were able to compensate for the combined stressor effects within six weeks. Nucleus colonies exposed to the combined stressors showed a short-term response with a less favorable brood to bee ratio and reduced colony development in May. No further impacts were observed in either the nucleus colonies or the full-sized colonies from July until the following spring. In addition, no fungicide-dependent differences were found in core gut and nectar microbiomes, and these differences were not distinguishable from local or environmental effects. Therefore, the provision of sufficient resources is important to increase the resilience of honey bees to a combination of stressors.

Gas Transport Mechanisms through Molecular Thin Carbon Nanomembranes.

Molecular thin carbon nanomembranes (CNMs) synthesized by electron irradiation induced cross-linking of aromatic self-assembled monolayers (SAMs) are promising 2D materials for the next generation of filtration technologies. Their unique properties including ultimately low thickness of ≈1 nm, sub-nanometer porosity, mechanical and chemical stability are attractive for the development of innovative filters with low energy consumption, improved selectivity, and robustness. However, the permeation mechanisms through CNMs resulting in, e.g., an ≈1000 times higher fluxes of water in comparison to helium have not been yet understood. Here, a study of the permeation of He, Ne, D2 , CO2 , Ar, O2 and D2 O using mass spectrometry in the temperature range from room temperature to ≈120 °C is studied. As a model system, CNMs made from [1″,4',1',1]-terphenyl-4-thiol SAMs are investigated. It is found out that all studied gases experience an activation energy barrier upon the permeation which scales with their kinetic diameters. Moreover, their permeation rates are dependent on the adsorption on the nanomembrane surface. These findings enable to rationalize the permeation mechanisms and establish a model, which paves the way toward the rational design not only of CNMs but also of other organic and inorganic 2D materials for energy-efficient and highly selective filtration applications.

[4.3.1]Bicyclic FKBP Ligands Inhibit Legionella Pneumophila Infection by LpMip-Dependent and LpMip-Independent Mechanisms.

Legionella pneumophila is the causative agent of Legionnaires' disease, a serious form of pneumonia. Its macrophage infectivity potentiator (Mip), a member of a highly conserved family of FK506-binding proteins (FKBPs), plays a major role in the proliferation of the gram-negative bacterium in host organisms. In this work, we test our library of >1000 FKBP-focused ligands for inhibition of LpMip. The [4.3.1]-bicyclic sulfonamide turned out as a highly preferred scaffold and provided the most potent LpMip inhibitors known so far. Selected compounds were non-toxic to human cells, displayed antibacterial activity and block bacterial proliferation in cellular infection-assays as well as infectivity in human lung tissue explants. The results confirm [4.3.1]-bicyclic sulfonamides as anti-legionellal agents, although their anti-infective properties cannot be explained by inhibition of LpMip alone.

Legionella pneumophila PPIase Mip Interacts with the Bacterial Proteins SspB, Lpc2061, and FlaA and Promotes Flagellation.

The peptidyl-prolyl-cis/trans-isomerase (PPIase) macrophage infectivity potentiator (Mip) contributes to the pathogenicity and fitness of L. pneumophila, the causative agent of Legionnaires' disease. Here, we identified the stringent starvation protein SspB, hypothetical protein Lpc2061, and flagellin FlaA as bacterial interaction partners of Mip. The macrolide FK506, which inhibits the PPIase activity of Mip, interfered with the binding of Lpc2061. Moreover, we demonstrated that the N-terminal dimerization region and amino acid Y185 in the C-terminal PPIase domain of Mip are required for the binding of Lpc2061 and FlaA. The modeling of the interaction partners and global docking with Mip suggested nonoverlapping binding interfaces, and a molecular dynamic simulation predicted an increased stability for the tripartite interaction of Lpc2061, Mip, and FlaA. On the functional level, we demonstrated that Mip promotes L. pneumophila flagellation, which is positively influenced by the binding of Lpc2061 and reduced by FK506. Also, L. pneumophila mutants expressing the Y185A or the monomeric Mip variant, which bind less Lpc2061, were nonmotile, were less flagellated, and yielded less FlaA when quantified. To our knowledge, this is the first report in which a PPIase and its bacterial interaction partners were demonstrated to influence flagellation.

Simultaneous Dimensional and Analytical Characterization of Ordered Nanostructures.

The spatial and compositional complexity of 3D structures employed in today's nanotechnologies has developed to a level at which the requirements for process development and control can no longer fully be met by existing metrology techniques. For instance, buried parts in stratified nanostructures, which are often crucial for device functionality, can only be probed in a destructive manner in few locations as many existing nondestructive techniques only probe the objects surfaces. Here, it is demonstrated that grazing exit X-ray fluorescence can simultaneously characterize an ensemble of regularly ordered nanostructures simultaneously with respect to their dimensional properties and their elemental composition. This technique is nondestructive and compatible to typically sized test fields, allowing the same array of structures to be studied by other techniques. For crucial parameters, the technique provides sub-nm discrimination capabilities and it does not require access-limited large-scale research facilities as it is compatible to laboratory-scale instrumentation.

Zinc metalloprotease ProA of Legionella pneumophila increases alveolar septal thickness in human lung tissue explants by collagen IV degradation.

ProA is a secreted zinc metalloprotease of Legionella pneumophila causing lung damage in animal models of Legionnaires' disease. Here we demonstrate that ProA promotes infection of human lung tissue explants (HLTEs) and dissect the contribution to cell type specific replication and extracellular virulence mechanisms. For the first time, we reveal that co-incubation of HLTEs with purified ProA causes a significant increase of the alveolar septal thickness. This destruction of connective tissue fibres was further substantiated by collagen IV degradation assays. The moderate attenuation of a proA-negative mutant in A549 epithelial cells and THP-1 macrophages suggests that effects of ProA in tissue mainly result from extracellular activity. Correspondingly, ProA contributes to dissemination and serum resistance of the pathogen, which further expands the versatile substrate spectrum of this thermolysin-like protease. The crystal structure of ProA at 1.48 Å resolution showed high congruence to pseudolysin of Pseudomonas aeruginosa, but revealed deviations in flexible loops, the substrate binding pocket S1 ' and the repertoire of cofactors, by which ProA can be distinguished from respective homologues. In sum, this work specified virulence features of ProA at different organisational levels by zooming in from histopathological effects in human lung tissue to atomic details of the protease substrate determination.

Heat stress response in the closest algal relatives of land plants reveals conserved stress signaling circuits.

All land plants (embryophytes) share a common ancestor that likely evolved from a filamentous freshwater alga. Elucidating the transition from algae to embryophytes - and the eventual conquering of Earth's surface - is one of the most fundamental questions in plant evolutionary biology. Here, we investigated one of the organismal properties that might have enabled this transition: resistance to drastic temperature shifts. We explored the effect of heat stress in Mougeotia and Spirogyra, two representatives of Zygnematophyceae - the closest known algal sister lineage to land plants. Heat stress induced pronounced phenotypic alterations in their plastids, and high-performance liquid chromatography-tandem mass spectroscopy-based profiling of 565 transitions for the analysis of main central metabolites revealed significant shifts in 43 compounds. We also analyzed the global differential gene expression responses triggered by heat, generating 92.8 Gbp of sequence data and assembling a combined set of 8905 well-expressed genes. Each organism had its own distinct gene expression profile; less than one-half of their shared genes showed concordant gene expression trends. We nevertheless detected common signature responses to heat such as elevated transcript levels for molecular chaperones, thylakoid components, and - corroborating our metabolomic data - amino acid metabolism. We also uncovered the heat-stress responsiveness of genes for phosphorelay-based signal transduction that links environmental cues, calcium signatures and plastid biology. Our data allow us to infer the molecular heat stress response that the earliest land plants might have used when facing the rapidly shifting temperature conditions of the terrestrial habitat.

Experimental observation of the short-range surface plasmon polariton mode and its longitudinal adiabatic compression in a metallic wedge.

In this study, we explore analytically and experimentally long- and short-range surface plasmon polariton (LR-SPP and SR-SPP, respectively) modes in gold wedges. Especially, we aim to observe the 2-dimensional confinement of the electromagnetic field in gold wedges as it could enhance the light-matter interaction by offering a local density of states which depends on the propagation constant, consequently on the wedge height. The LR-SPP mode can propagate over a long distance, but the real part of the propagation constant remains relatively insensitive to the decreasing wedge height. This mode also experiences cut-off at a wedge height of about 50 nm in our experimental condition. Meanwhile, the SR-SPP mode has a large propagation constant that increases further with decreasing wedge height. As a result, the effective wavelength of the mode shrinks confining the electromagnetic wave longitudinally along the propagation direction in addition to enhancing the transverse confinement of SR-SPP. In the experiment, we use gold wedges with different edge heights to excite each SPP mode individually and image the electromagnetic near field by using a pseudo-heterodyne scattering scanning near-field optical microscope. By imaging the LR-SPP mode field, we demonstrate that the theoretical and measured values of the effective wavelength agree quite well. By using short wedges, we measure the SR-SPP mode field and demonstrate that the effective wavelength decreases to 47% in about half a micrometer of propagation distance. This corresponds to a 3.5 times decrease of the vacuum wavelength or an effective index of 3.5. It is important to note that this value is, by no means, the limit of the electromagnetic field's longitudinal confinement in a gold wedge. Rather, we were only able to measure the electromagnetic field up to this point due to our measurement limitations. The electromagnetic field will be propagating further, and the longitudinal confinement will increase as well. In conclusion, we measured the SR-SPP in a gold wedge and demonstrate the electromagnetic field confinement in the visible spectrum in gold wedges.

Streptomonospora litoralis sp. nov., a halophilic thiopeptides producer isolated from sand collected at Cuxhaven beach.

Strain M2T was isolated from the beach of Cuxhaven, Wadden Sea, Germany, in course of a program to attain new producers of bioactive natural products. Strain M2T produces litoralimycin and sulfomycin-type thiopeptides. Bioinformatic analysis revealed a potential biosynthetic gene cluster encoding for the M2T thiopeptides. The strain is Gram-stain-positive, rod shaped, non-motile, spore forming, showing a yellow colony color and forms extensively branched substrate mycelium and aerial hyphae. Inferred from the 16S rRNA gene phylogeny strain M2T affiliates with the genus Streptomonospora. It shows 96.6% 16S rRNA gene sequence similarity to the type species Streptomonospora salina DSM 44593 T and forms a distinct branch with Streptomonospora sediminis DSM 45723 T with 97.0% 16S rRNA gene sequence similarity. Genome-based phylogenetic analysis revealed that M2T is closely related to Streptomonospora alba YIM 90003 T with a digital DNA-DNA hybridisation (dDDH) value of 26.6%. The predominant menaquinones of M2T are MK-10(H6), MK-10(H8), and MK-11(H6) (> 10%). Major cellular fatty acids are iso-C16:0, anteiso C17:0 and C18:0 10-methyl. The polar lipid profile consisted of diphosphatidylglycerol phosphatidyl glycerol, phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, three glycolipids, two unknown phospholipids, and two unknown lipids. The genome size of type strain M2T is 5,878,427 bp with 72.1 mol % G + C content. Based on the results obtained from phylogenetic and chemotaxonomic studies, strain M2T (= DSM 106425 T = NCCB 100650 T) is considered to represent a novel species within the genus Streptomonospora for which the name Streptomonospora litoralis sp. nov. is proposed.

Second-Harmonic Generation in Resonant Nonlinear Metasurfaces Based on Lithium Niobate.

Lithium niobate is an excellent and widely used material for nonlinear frequency conversion due to its strong optical nonlinearity and broad transparency region. Here, we report the fabrication and experimental investigation of resonant nonlinear metasurfaces for second-harmonic generation based on thin-film lithium niobate. In the fabricated metasurfaces, we observe pronounced Mie-type resonances leading to enhanced second-harmonic generation in the direction normal to the metasurface. We find the largest second-harmonic generation efficiency for the resonance dominated by the electric contributions because its specific field distribution enables the most efficient usage of the largest element of the lithium niobate nonlinear susceptibility tensor. This is confirmed by polarization-resolved second-harmonic measurements, where we study contributions from different elements of the nonlinear susceptibility tensor to the total second-harmonic signal. Our work facilitates establishing lithium niobate as a material for resonant nanophotonics.

Discovery of Paenibacillus larvae ERIC V: Phenotypic and genomic comparison to genotypes ERIC I-IV reveal different inventories of virulence factors which correlate with epidemiological prevalences of American Foulbrood.

Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a highly contagious brood disease of honey bees (Apis mellifera). AFB requires mandatory reporting to the veterinary authority in many countries and until now four genotypes, P. larvae ERIC I-IV, have been identified. We isolated a new genotype, ERIC V, from a Spanish honey sample. After a detailed phenotypic comparison with the reference strains of the ERIC I-IV genotypes, including spore morphology, non-ribosomal peptide (NRP) profiling, and in vivo infections of A. mellifera larvae, we established a genomic DNA Macrorestriction Fragment Pattern Analysis (MRFPA) scheme for future epidemiologic discrimination. Whole genome comparison of the reference strains and the new ERIC V genotype (DSM 106052) revealed that the respective virulence gene inventories of the five genotypes corresponded with the time needed to kill 100 % of the infected bee larvae (LT100) in in vivo infection assays. The rarely isolated P. larvae genotypes ERIC II I-V with a fast-killing phenotype (LT100 3 days) harbor genes with high homology to virulence factors of other insect pathogens. These virulence genes are absent in the epidemiologically prevalent genotypes ERIC I (LT100 12 days) and ERIC II (LT100 7 days), which exhibit slower killing phenotypes. Since killing-retardation is known to reduce the success of hygienic cleaning by nurse bees, the identified absence of virulence factors might explain the epidemiological prevalences of ERIC genotypes. The discovery of the P. larvae ERIC V isolate suggests that more unknown ERIC genotypes exist in bee colonies. Since inactivation or loss of a few genes can transform a fast-killing phenotype into a more dangerous slow-killing phenotype, these rarely isolated genotypes may represent a hidden reservoir for future AFB outbreaks.

Laser-induced spatially-selective tailoring of high-index dielectric metasurfaces.

Optically resonant high-index dielectric metasurfaces featuring Mie-type electric and magnetic resonances are usually fabricated by means of planar technologies, which limit the degrees of freedom in tunability and scalability of the fabricated systems. Therefore, we propose a complimentary post-processing technique based on ultrashort (≤ 10 ps) laser pulses. The process involves thermal effects: crystallization and reshaping, while the heat is localized by a high-precision positioning of the focused laser beam. Moreover, for the first time, the resonant behavior of dielectric metasurface elements is exploited to engineer a specific absorption profile, which leads to a spatially-selective heating and a customized modification. Such technique has the potential to reduce the complexity in the fabrication of non-uniform metasurface-based optical elements. Two distinct cases, a spatial pixelation of a large-scale metasurface and a height modification of metasurface elements, are explicitly demonstrated.

Single-shot characterization of strongly focused coherent XUV and soft X-ray beams.

In this Letter, we present a novel, to the best of our knowledge, single-shot method for characterizing focused coherent beams. We utilize a dedicated amplitude-only mask, in combination with an iterative phase retrieval algorithm, to reconstruct the amplitude and phase of a focused beam from a single measured far-field diffraction pattern alone. In a proof-of-principle experiment at a wavelength of 13.5 nm, we demonstrate our new method and obtain an RMS phase error of better than λ/70. This method will find applications in the alignment of complex optical systems, real-time feedback to adaptive optics, and single-shot beam characterization, e.g., at free-electron lasers or high-order harmonic beamlines.

Manipulation of Magnetic Dipole Emission from Eu3+ with Mie-Resonant Dielectric Metasurfaces.

Mie-resonant high-index dielectric nanoparticles and metasurfaces have been suggested as a viable platform for enhancing both electric and magnetic dipole transitions of fluorescent emitters. While the enhancement of the electric dipole transitions by such dielectric nanoparticles has been demonstrated experimentally, the case of magnetic-dipole transitions remains largely unexplored. Here, we study the enhancement of spontaneous emission of Eu3+ ions, featuring both electric and magnetic-dominated dipole transitions, by dielectric metasurfaces composed of Mie-resonant silicon nanocylinders. By coating the metasurfaces with a layer of an Eu3+ doped polymer, we observe an enhancement of the Eu3+ emission associated with the electric (at 610 nm) and magnetic-dominated (at 590 nm) dipole transitions. The enhancement factor depends systematically on the spectral proximity of the atomic transitions to the Mie resonances as well as their multipolar order, both controlled by the nanocylinder size. Importantly, the branching ratio of emission via the electric or magnetic transition channel can be modified by carefully designing the metasurface, where the magnetic dipole transition is enhanced more than the electric transition for cylinders with radii of about 130 nm. We confirm our observations by numerical simulations based on the reciprocity principle. Our results open new opportunities for bright nanoscale light sources based on magnetic transitions.

Controlling second-harmonic diffraction by nano-patterning MoS2 monolayers.

Monolayers of transition metal dichalcogenides have a strong second-order nonlinear response enabling second-harmonic generation. Here, we control the spatial radiation properties of the generated second harmonic by patterning MoS2 monolayers using focused ion beam milling. We observe diffraction of the second harmonic into the zero and first diffraction orders via an inscribed one-dimensional grating. Additionally, we included a fork-like singularity into the grating to create a vortex beam in the first diffraction order.

FKBPs in bacterial infections.

BACKGROUND: FK506-binding proteins (FKBPs) contain a domain with peptidyl-prolyl-cis/trans-isomerase (PPIase) activity and bind the immunosuppressive drugs FK506 and rapamycin. FKBPs belong to the immunophilin family and are found in eukaryotes and bacteria. SCOPE OF REVIEW: In this review we describe two major groups of bacterial virulence-associated FKBPs, the trigger factor and Mip-like PPIases. Moreover, we discuss the contribution of host FKBPs in bacterial infection processes. MAJOR CONCLUSIONS: Since PPIases are regarded as alternative antiinfective drug targets we highlight current research strategies utilizing pipecolinic acid and cycloheximide derivatives as well as substrate based inhibitors. GENERAL SIGNIFICANCE: The current research strategies suggest a beneficial synergism of drug development and basic research. This article is part of a Special Issue entitled Proline-directed Foldases: Cell Signaling Catalysts and Drug Targets.