A GFP-expressing minigenome of a chrysovirus replicating in fungi.

The Fusarium graminearum virus China 9 (FgV-ch9) is a member of the genus Betachrysovirus in the Chrysoviridae family and causes hypovirulence in its host, Fusarium graminearum, the causal agent of Fusarium head blight. Although insights into viral biology of FgV-ch9 have expanded in recent years, questions regarding the function of virus-encoded proteins, cis-acting elements, and virus transmission are yet to be answered. Therefore, we developed a tool for the establishment of an artificial 6th segment of FgV-ch9, which encodes a GFP gene flanked by the non-translated regions of FgV-ch9 segment 1. Subsequently, we have proved successful encapsidation of this artificial segment into virus particles as well as its horizontal transmission. Expression of GFP was further verified via immunoassay and life cell imaging. Thus far, we were able to establish for the first time a mini-replicon system for segmented dsRNA viruses replicating in fungi.

A virus from Aspergillus cibarius with features of alpha- and betachrysoviruses.

From the ascomycete Aspergillus cibarius strain NW-FVA 2590, which was originally isolated from a root, associated with stem collar necrosis of Fraxinus excelsior L., a novel virus was isolated and characterized. Its genome is encoded on three monocistronic dsRNA segments ranging from 3683 bp (dsRNA 1) over 3093 (dsRNA 2) to 2902 bp (dsRNA 3), which are packed in isometric particles of around 35 nm. While the viral RdRp (P1) is encoded on segment 1, protein sequencing showed that two more structural proteins are present which are translated from dsRNA 2 (P2) and dsRNA 3 (P3) and possibly form the viral capsid. Additionally, P2 and P3 may undergo posttranslational modifications since the detected proteins bands deviated from the calculated sizes. Due to its phylogenetic position, the novel virus was grouped in the family of Chrysoviridae and was tentatively denominated as Aspergillus cibarius chrysovirus 1 (AcCV1). Due to its composition, biological properties and phylogenetic position, distant from the genera Alphachrysovirus and Betachrysovirus, we suggest to position AcCV1 in a proposed genus "Gammachrysovirus".

Fast preparation of high-quality viral dsRNA from fungal tissue by commercial nucleic acid extraction kits.

The genomes of most known mycoviruses consist of double stranded RNA (dsRNA) or single stranded RNA (ssRNA). Therefore, for all aspects of mycovirology, the research is highly dependent on the quality and quantity of RNA either by the extraction of genomic dsRNA or dsRNA as a replicating intermediate. A common procedure to extract dsRNA is its binding on a cellulose matrix after a phenol/chloroform purification step. A commercial kit for dsRNA extraction facilitated the researchers´ daily work, but is not available anymore. To extract nucleic acids in a standardized good quality and quantity from small amounts of starting material, we compared commercial kits for gDNA extraction to the kits for RNA extraction using fungal material with a high and a low virus titer. Here we show that viral dsRNA can be extracted using commercial gDNA kits from fungal tissue with a high and a low virus titer in the same quality and quantity as it was done with the discontinued dsRNA extraction kit.

[The complexity of hand-surgical procedures in the context of cross-sector care concepts: a consensus recommendation of the German Society of Hand Surgery] / Die Komplexität handchirurgischer Therapien im Kontext sektorenübergreifender Versorgungskonzepte: Konsensusempfehlung der Deutschen Gesellschaft für Handchirurgie.

Overboarding politcal influence in Germany concerning medical issues has come to a new peak. The report by the IGES Institute published in 2022 made an important contribution in this regard. Unfortunately, only that part of the recommendations of this report were implemented in a new version of the contract for outpatient surgery according to Section 115b SGB V (AOP contract), that called for an expansion of outpatient surgery. In particular, those aspects that are important from a medical point of view for a patient-specific adjustment of outpatient surgery (e. g. old age, frailty, comorbidities) as well as the important structural requirements for outpatient postoperative care were included in the new AOP contract at best in a rudimentary manner. For this reason, the German Society for Hand Surgery felt compelled to give its members a recommendation as to which medical aspects must be taken into account, especially when performing hand surgery operations, in order to ensure the highest level of safety for the patients entrusted to us while performing outpatient surgery. An expert group of experienced hand surgeons and hand therapists who work in hospitals of all levels of care as well as resident surgeons was formed in order to formulate mutually agreed recommendations for action.

Complete genome sequence of a novel alternavirus infecting the fungus Ilyonectria crassa.

A novel dsRNA mycovirus named Ilyonectria crassa alternavirus 1 (IcAV1) was found in Ilyonectria crassa isolate NW-FVA 1829. The fungus was isolated from an ash (Fraxinus excelsior L.) necrotic trunk disc infected with Hymenoscyphus fraxineus [(T. Kowalski) Baral, Queloz, Hosoya] causing ash dieback. The complete genome of IcAV1 is composed of three segments, each containing a single ORF on the positive-sense RNA. The extreme 5' UTRs of dsRNA 1 (3604 bp), dsRNA 2 (2547 bp), and dsRNA 3 (2518 bp) share a conserved hexadecamer sequence (5'-GGCTGTGTGTTTAGTT-3') and are capped. The 3' UTRs are polyadenylated. In silico analysis showed that the viral RdRP is encoded on dsRNA 1 and the capsid-protein subunits are encoded on dsRNA 3. Maximum-likelihood analysis of the aa sequence of the viral RdRP showed that IcAV1 clusters with alternaviruses from Fusarium spp., while the type member of the proposed family "Alternaviridae", Alternaria alternata virus 1 (AaV1), formed a clade together with Stemphylium lycopersici mycovirus (SlV). The function of the protein encoded on segment 2 is unknown. Based on its genome organization and its phylogenetic position, IcAV1 is suggested to be a new member of the proposed family "Alternaviridae". This is the first report of a mycovirus infecting I. crassa.

Stable overexpression and targeted gene deletion of the causative agent of ash dieback Hymenoscyphus fraxineus.

BACKGROUND: Due to the infection with the invasive ascomycete Hymenoscyphus fraxineus, which has been replacing the closely related and non-pathogenic native Hymenoscyphus albidus, the European ashes, Fraxinus excelsior (also known as the common ash), Fraxinus angustifolia (also known as narrow-leaved ash) and Fraxinus ornus (also known as the manna ash) are at risk. Hymenoscyphus fraxineus is the causative agent of ash dieback of the European ashes, but is non-pathogenic to the native Asian ash Fraxinus mandshurica (also known as the Manchurian ash). Even though the invasion of H. fraxineus is a great threat for ashes in Europe, the fungal biology is still poorly understood. By the use of live cell imaging and targeted gene knock-out, the fungal life cycle and host-pathogen interaction can be studied in more detail. RESULTS: Here, we developed a protocol for the preparation of protoplasts from mycelium of H. fraxineus, for their regeneration and for stable transformation with reporter genes and targeted gene knock-out by homologous recombination. We obtained mutants with various levels of reporter gene expression which did not correlate with the number of integrations. In an in vitro infection assay, we demonstrated the suitability of reporter gene overexpression for fungal detection in plant tissue after inoculation. As a proof of principle for targeted gene knock-out, the hygromycin resistance cassette of a reporter gene-expressing mutant was replaced with a geneticin resistance cassette. CONCLUSIONS: The invasive fungal pathogen H. fraxineus is threatening the European ashes. To develop strategies for pest management, a better understanding of the fungal life cycle and its host interaction is crucial. Here, we provide a protocol for stable transformation of H. fraxineus to obtain fluorescence reporter strains and targeted gene knock-out mutants. This protocol will help future investigations on the biology of this pathogen.

Characterization of a novel alternavirus infecting the fungal pathogen Fusarium solani.

A novel dsRNA mycovirus was found in Fusarium solani (F. solani) strain NW-FVA 2572. The fungus was originally isolated from a root, associated with stem collar necrosis of Fraxinus excelsior L. The viral genome is composed of four segments, which range from around 3.5 kbp to 1.7 kbp (RNA 1: 3522 bp; RNA 2: 2633 bp; RNA 3: 2403 bp; RNA 4: 1721 bp). The segments share a conserved and capped 5'-terminus and their 3'-termini are polyadenylated. Protein sequencing showed that the viral RdRP is encoded on segment 1. The virus clusters together with Aspergillus mycovirus 341 (AsV341), Aspergillus heteromorphus alternavirus 1 (AheAV1), Aspergillus foetidus virus-fast (AfV-F) and Cordyceps chanhua alternavirus 1 (CcAV1). As highest value, the RdRP showed 61.50% identical amino acids with P1 of the AfV-F. The capsid protein is encoded on segment 3, the proteins encoded on RNA 2 and RNA 4 are of unknown function. Segment 4 harbors large UTRs (186 nts at the 5'-terminus and 311 nts at the 3'-terminus). Based on its genome organization and phylogenetic position, the virus is suggested to be a new member of the proposed family Alternaviridae and was therefore named Fusarium solani alternavirus 1 (FsAV1). This is the first report of an Alternavirus infecting a fungus of the F. solani species complex (FSSC).

Complete genome sequence of a new quadrivirus infecting a member of the genus Thelonectria.

A novel dsRNA virus named "Thelonectria quadrivirus 1" (TQV1) was found in a member of the genus Thelonectria (Ascomycota), isolated from a root associated with stem collar necrosis of Fraxinus excelsior L. The complete genome of TQV1 is composed of four segments, each containing a single ORF on the positive sense RNA. The sequence of the 5´ (5´-(C/T)ACGAAAAA-3´) and 3´termini (5´AT(T/G)AGCAATG(T/C)GC(G/A)CG-3') of dsRNA 1 (4876 bp), dsRNA 2 (4312 bp), dsRNA 3 (4158 bp), and dsRNA 4 (3933 bp) are conserved. Based on its genome organization and phylogenetic position, TQV1 is suggested to be a new member of the family Quadriviridae. This is the first report of a mycovirus infecting a member of the genus Thelonectria.

Processing of the capsid proteins of the Betachrysovirus Fusarium graminearum virus-China 9 (FgV-ch9).

While the capsid of viruses in the Alphachrysovirus genus is built of subunits of a single coat protein, the capsid of viruses grouped in the Betachrysovirus genus may consist of subunits of two different proteins. For four of these betachrysoviruses, the detected molecular weights of the putative coat proteins differ from the sizes deduced from the nucleic acid sequence. The origin of these modifications remained unclear and it was hypothesized that the coat proteins undergo unspecific degradation. In our study, we show that these modifications are based on processing steps performed by unknown factors present in extracts of several eukaryotic organisms. Furthermore, we show that the C-terminal domain of P3 is fully degraded after capsid processing and particle assembly.

Repeat DNA-PAINT suppresses background and non-specific signals in optical nanoscopy.

DNA-PAINT is a versatile optical super-resolution technique relying on the transient binding of fluorescent DNA 'imagers' to target epitopes. Its performance in biological samples is often constrained by strong background signals and non-specific binding events, both exacerbated by high imager concentrations. Here we describe Repeat DNA-PAINT, a method that enables a substantial reduction in imager concentration, thus suppressing spurious signals. Additionally, Repeat DNA-PAINT reduces photoinduced target-site loss and can accelerate sampling, all without affecting spatial resolution.

Detecting Nanoscale Distribution of Protein Pairs by Proximity-Dependent Super-resolution Microscopy.

Interactions between biomolecules such as proteins underlie most cellular processes. It is crucial to visualize these molecular-interaction complexes directly within the cell, to show precisely where these interactions occur and thus improve our understanding of cellular regulation. Currently available proximity-sensitive assays for in situ imaging of such interactions produce diffraction-limited signals and therefore preclude information on the nanometer-scale distribution of interaction complexes. By contrast, optical super-resolution imaging provides information about molecular distributions with nanometer resolution, which has greatly advanced our understanding of cell biology. However, current co-localization analysis of super-resolution fluorescence imaging is prone to false positive signals as the detection of protein proximity is directly dependent on the local optical resolution. Here we present proximity-dependent PAINT (PD-PAINT), a method for subdiffraction imaging of protein pairs, in which proximity detection is decoupled from optical resolution. Proximity is detected via the highly distance-dependent interaction of two DNA constructs anchored to the target species. Labeled protein pairs are then imaged with high-contrast and nanoscale resolution using the super-resolution approach of DNA-PAINT. The mechanisms underlying the new technique are analyzed by means of coarse-grained molecular simulations and experimentally demonstrated by imaging DNA-origami tiles and epitopes of cardiac proteins in isolated cardiomyocytes. We show that PD-PAINT can be straightforwardly integrated in a multiplexed super-resolution imaging protocol and benefits from advantages of DNA-based super-resolution localization microscopy, such as high specificity, high resolution, and the ability to image quantitatively.

3D super-resolution microscopy performance and quantitative analysis assessment using DNA-PAINT and DNA origami test samples.

Assessment of the imaging quality in localisation-based super-resolution techniques relies on an accurate characterisation of the imaging setup and analysis procedures. Test samples can provide regular feedback on system performance and facilitate the implementation of new methods. While multiple test samples for regular, 2D imaging are available, they are not common for more specialised imaging modes. Here, we analyse robust test samples for 3D and quantitative super-resolution imaging, which are straightforward to use, are time- and cost-effective and do not require experience beyond basic laboratory and imaging skills. We present two options for assessment of 3D imaging quality, the use of microspheres functionalised for DNA-PAINT and a commercial DNA origami sample. A method to establish and assess a qPAINT workflow for quantitative imaging is demonstrated with a second, commercially available DNA origami sample.

True Molecular Scale Visualization of Variable Clustering Properties of Ryanodine Receptors.

Signaling nanodomains rely on spatial organization of proteins to allow controlled intracellular signaling. Examples include calcium release sites of cardiomyocytes where ryanodine receptors (RyRs) are clustered with their molecular partners. Localization microscopy has been crucial to visualizing these nanodomains but has been limited by brightness of markers, restricting the resolution and quantification of individual proteins clustered within. Harnessing the remarkable localization precision of DNA-PAINT (<10 nm), we visualized punctate labeling within these nanodomains, confirmed as single RyRs. RyR positions within sub-plasmalemmal nanodomains revealed how they are organized randomly into irregular clustering patterns leaving significant gaps occupied by accessory or regulatory proteins. RyR-inhibiting protein junctophilin-2 appeared highly concentrated adjacent to RyR channels. Analyzing these molecular maps showed significant variations in the co-clustering stoichiometry between junctophilin-2 and RyR, even between nearby nanodomains. This constitutes an additional level of complexity in RyR arrangement and regulation of calcium signaling, intrinsically built into the nanodomains.

isoSTED nanoscopy with intrinsic beam alignment.

Despite the need for isotropic optical resolution in a growing number of applications, the majority of super-resolution fluorescence microscopy setups still do not attain an axial resolution comparable to that in the lateral dimensions. Three-dimensional (3D) nanoscopy implementations that employ only a single objective lens typically feature a trade-off between axial and lateral resolution. 4Pi arrangements, in which the sample is illuminated coherently through two opposing lenses, have proven their potential for rendering the resolution isotropic. However, instrument complexity due to a large number of alignment parameters has so far thwarted the dissemination of this approach. Here, we present a 4Pi-STED setup combination, also called isoSTED nanoscope, where the STED and excitation beams are intrinsically co-aligned. A highly robust and convenient 4Pi cavity allows easy handling without the need for readjustments during imaging experiments.

TIPS-tetracene- and TIPS-pentacene-annulated poly(norbornadiene)s: synthesis and properties.

The synthesis of tetracene- and pentacene-annulated norbornadienes, formed through the Diels-Alder reaction of a dehydroacene with cyclopentadiene is reported. Ring-opening metathesis polymerization (ROMP) leads to polymers that are investigated with respect to their physical, optical, and electronic properties by gel permeation chromatography (GPC), UV-vis spectroscopy, and cyclic voltammetry. The pentacene-containing polymer P1 is successfully integrated into an organic field-effect transistor (OFET); the tetracene-containing polymer P2 is integrated into an organic light-emitting diode (OLED).