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A global existential and interlocking environmental, climate and cooperation/equity polycrisis is being faced, which increasingly impacts and is impacted by water and land systems. As a result, transformations in response are gaining increasing traction. Advances in approaches to visualising and communicating how innovations and changes in landscape features enable shifts, transformations and transitions are more crucial than ever before. Visions help focus the actions, collaboration and alignment of multiple actors in working towards a common purpose, whilst also entering people’s consciousness at the deep level of values, transforming beliefs and consequently, thinking and action. They give direction to effort and pull on transformational innovation. The evidence-based ‘Water Visions Visualisation Platform’ presents an innovative and accessible way to illustrate, communicate and support future water visioning and strategising at the landscape scale based on composite paradigms, scenarios, horizons and concepts. Plausible visions of the future are envisioned, illustrated, narratively described and qualitatively assessed, as well as connected with real-world examples and resources through the interactive platform. This paper outlines the co-creation methodology, the architecture and the initial co-development of the platform, as well as a preliminary evaluation of its efficacy through literature-based criteria and the sharing of the platform with stakeholders.

Elissa Burton,1,2 Vinicius Cavalheri,1 Richard Adams,3 Colleen Oakley Browne,4 Petra Bovery-Spencer,4 Audra M Fenton,3 Bruce W Campbell,5 Keith D Hill1,6 1School of Physiotherapy and Exercise Science, Curtin University, Perth, WA, Australia; 2Research Department, Silver Chain, Perth, WA, Australia; 3Community Services, West Gippsland Healthcare Group, Warragul, VIC, Australia; 4Falls Prevention for People Living with Dementia Project, Central West Gippsland Primary Care Partnership, Moe, VIC, Australia; 5Allied Health, Latrobe Regional Hospital, Traralgon, VIC, Australia; 6Preventive and Public Health Division, National Ageing Research Institute, Melbourne, VIC, Australia Objective: The objective of this systematic review and meta-analysis is to evaluate the effectiveness of exercise programs to reduce falls in older people with dementia who are living in the community.Method: Peer-reviewed articles (randomized controlled trials [RCTs] and quasi-experimental trials) published in English between January 2000 and February 2014, retrieved from six electronic databases – Medline (ProQuest), CINAHL, PubMed, PsycInfo, EMBASE and Scopus – according to predefined inclusion criteria were included. Where possible, results were pooled and meta-analysis was conducted.Results: Four articles (three RCT and one single-group pre- and post-test pilot study) were included. The study quality of the three RCTs was high; however, measurement outcomes, interventions, and follow-up time periods differed across studies. On completion of the intervention period, the mean number of falls was lower in the exercise group compared to the control group (mean difference [MD] [95% confidence interval {CI}] =-1.06 [-1.67 to -0.46] falls). Importantly, the exercise intervention reduced the risk of being a faller by 32% (risk ratio [95% CI] =0.68 [0.55–0.85]). Only two other outcomes were reported in two or more of the studies (step test and physiological profile assessment). No between-group differences were observed in the results of the step test (number of steps) (MD [95% CI] =0.51 [-1.77 to 2.78]) or the physiological profile assessment (MD [95% CI] =-0.10 [-0.62 to 0.42]).Conclusion: Findings from this review suggest that an exercise program may potentially assist in preventing falls of older people with dementia living in the community. However, further research is needed with studies using larger sample sizes, standardized measurement outcomes, and longer follow-up periods, to inform evidence-based recommendations. Keywords: cognitive impairment, older people, physical activity, fallers, community dwelling

Abstract The genomic DNA sequence of a novel enteric uncultured microphage, ΦCA82 from a turkey gastrointestinal system was determined utilizing metagenomics techniques. The entire circular, single-stranded nucleotide sequence of the genome was 5,514 nucleotides. The ΦCA82 genome is quite different from other microviruses as indicated by comparisons of nucleotide similarity, predicted protein similarity, and functional classifications. Only three genes showed significant similarity to microviral proteins as determined by local alignments using BLAST analysis. ORF1 encoded a predicted phage F capsid protein that was phylogenetically most similar to the Microviridae ΦMH2K member's major coat protein. The ΦCA82 genome also encoded a predicted minor capsid protein (ORF2) and putative replication initiation protein (ORF3) most similar to the microviral bacteriophage SpV4. The distant evolutionary relationship of ΦCA82 suggests that the divergence of this novel turkey microvirus from other microviruses may reflect unique evolutionary pressures encountered within the turkey gastrointestinal system.

Abstract Background Because biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen. Results Phage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase) and a holin (PF04531). Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1) strongly significant host-specific sequence variation within the endolysin, and 2) a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical. Conclusions Significant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products.

Abstract Background The origin and evolution of the homologous GTP-binding cytoskeletal proteins FtsZ typical of Bacteria and tubulin characteristic of eukaryotes is a major question in molecular evolutionary biology. Both FtsZ and tubulin are central to key cell biology processes – bacterial septation and cell division in the case of FtsZ and in the case of tubulins the function of microtubules necessary for mitosis and other key cytoskeleton-dependent processes in eukaryotes. The origin of tubulin in particular is of significance to models for eukaryote origins. Most members of domain Bacteria possess FtsZ, but bacteria in genus Prosthecobacter of the phylum Verrucomicrobia form a key exception, possessing tubulin homologs BtubA and BtubB. It is therefore of interest to know whether other members of phylum Verrucomicrobia possess FtsZ or tubulin as their FtsZ-tubulin gene family representative. Results Verrucomicrobium spinosum, a member of Phylum Verrucomicrobia of domain Bacteria, has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The deduced amino acid sequence has sequence signatures and predicted secondary structure characteristic for FtsZ rather than tubulin, but phylogenetic trees and sequence analysis indicate that it is divergent from all other known FtsZ sequences in members of domain Bacteria. The FtsZ gene of V. spinosum is located within a dcw gene cluster exhibiting gene order conservation known to contribute to the divisome in other Bacteria and comparable to these clusters in other Bacteria, suggesting a similar functional role. Conclusion Verrucomicrobium spinosum has been found to possess a gene for a protein homologous to the cytoskeletal protein FtsZ. The results suggest the functional as well as structural homology of the V. spinosum FtsZ to the FtsZs of other Bacteria implying its involvement in cell septum formation during division. Thus, both bacteria-like FtsZ and eukaryote-like tubulin cytoskeletal homologs occur in different species of the phylum Verrucomicrobia of domain Bacteria, a result with potential major implications for understanding evolution of tubulin-like cytoskeletal proteins and the origin of eukaryote tubulins.

Secondary metabolites are generally produced by enzymes encoded by genes within a biosynthetic gene cluster. Transcription factor genes are frequently located within these gene clusters. These transcription factors often drive expression of the other genes of the biosynthetic gene cluster, and overexpression of the transcription factor provides a facile approach to express all genes within a gene cluster, resulting in production of downstream metabolite(s). Unfortunately this approach is not always successful, leading us to engineer more effective hybrid transcription factors. Herein, we attempted to activate a putative cryptic biosynthetic gene cluster in Aspergillus nidulans using a combination of transcription factor engineering and overexpression approaches. This resulted in the discovery of a novel secondary metabolite we term triorsellinaldehyde. Surprisingly, deletion of the polyketide synthase gene within the gene cluster did not prevent triorsellinaldehyde production. However, targeted deletion of a polyketide synthase gene elsewhere in the genome revealed its role in triorsellinaldehyde biosynthesis.

Carbon fiber reinforced polymers (CFRPs, or composites) are increasingly replacing traditional manufacturing materials used in the automobile, aerospace, and energy sectors. With this shift, it is vital to develop end-of-life processes for CFRPs that retain the value of both the carbon fibers and the polymer matrix. Here we demonstrate a strategy to upcycle pre- and postconsumer polystyrene-containing CFRPs, cross-linked with unsaturated polyesters or vinyl esters, to benzoic acid. The thermoset matrix is upgraded via biocatalysis utilizing an engineered strain of the filamentous fungus Aspergillus nidulans , which gives access to valuable secondary metabolites in high yields, exemplified here by (2 Z ,4 Z ,6 E )-octa-2,4,6-trienoic acid. Reactions are engineered to preserve the carbon fibers with much of their sizing so that the isolated carbon fiber plies are manufactured into new composite coupons that exhibit mechanical properties comparable to those of virgin manufacturing substrates. In sum, this represents the first system to reclaim a high value from both the fiber fabric and polymer matrix of a CFRP.

Quorum sensing (QS) is a cell-cell signaling system that enables bacteria to coordinate population density-dependent changes in behavior. This chemical communication pathway is mediated by diffusible N -acyl L-homoserine lactone signals and cytoplasmic signal-responsive LuxR-type receptors in Gram-negative bacteria. As many common pathogenic bacteria use QS to regulate virulence, there is significant interest in disrupting QS as a potential therapeutic strategy. Prior studies have implicated the natural products salicylic acid, cinnamaldehyde, and other related benzaldehyde derivatives as inhibitors of QS in the opportunistic pathogen Pseudomonas aeruginosa, yet we lack an understanding of the mechanisms by which these compounds function. Herein, we evaluate the activity of a set of benzaldehyde derivatives using heterologous reporters of the P. aeruginosa LasR and RhlR QS signal receptors. We find that most tested benzaldehyde derivatives can antagonize LasR or RhlR reporter activation at micromolar concentrations, although certain molecules also cause mild growth defects and nonspecific reporter antagonism. Notably, several compounds showed promising RhlR or LasR-specific inhibitory activities over a range of concentrations below that causing toxicity. ortho -Vanillin, a previously untested compound, was the most promising within this set. Competition experiments against the native ligands for LasR and RhlR revealed that ortho -vanillin can interact competitively with RhlR but not with LasR. Overall, these studies expand our understanding of benzaldehyde activities in the LasR and RhlR receptors and reveal potentially promising effects of ortho -vanillin as a small molecule QS modulator against RhlR., Importance: Quorum sensing (QS) regulates many aspects of bacterial pathogenesis and has attracted much interest as a target for anti-virulence therapies over the past 30 years, for example, antagonists of the LasR and RhlR QS receptors in Pseudomonas aeruginosa . Potent and selective QS inhibitors remain relatively scarce. However, natural products have provided a bounty of chemical scaffolds with anti-QS activities, but their molecular mechanisms are poorly characterized. The current study serves to fill this void by examining the activity of an important and wide-spread class of natural product QS modulators, benzaldehydes, and related derivatives, in LasR and RhlR. We demonstrate that ortho -vanillin can act as a competitive inhibitor of RhlR, a receptor that has emerged and may supplant LasR in certain settings as a target for P. aeruginosa QS control. The results and insights provided herein will advance the design of chemical tools to study QS with improved activities and selectivities., Competing Interests: The authors declare no conflict of interest.

Fungal secondary metabolite (SM) biosynthetic gene clusters (BGCs) containing dimethylallyltryptophan synthases (DMATSs) produce structurally diverse prenylated indole alkaloids with wide-ranging activities that have vast potential as human therapeutics. To discover new natural products produced by DMATSs, we mined the Department of Energy Joint Genome Institute's MycoCosm database for DMATS-containing BGCs. We found a DMATS BGC in Aspergillus homomorphus CBS 101889, which also contains a nonribosomal peptide synthetase (NRPS). This BGC appeared to have a previously unreported combination of genes, which suggested the cluster might make novel SMs. We refactored this BGC with highly inducible promoters into the model fungus Aspergillus nidulans . The expression of this refactored BGC in A . nidulans resulted in the production of eight tryptophan-containing diketopiperazines, six of which are new to science. We have named them homomorphins A-F ( 2 , 4 - 8 ). Perhaps even more intriguingly, to our knowledge, this is the first discovery of C4-prenylated tryptophan-containing diketopiperazines and their derivatives. In addition, the NRPS from this BGC is the first described that has the ability to promiscuously combine tryptophan with either of two different amino acids, in this case, l-valine or l- allo -isoleucine.

Wild-type Aspergillus nidulans asexual spores (conidia) are green due to a pigment that protects the spores against ultraviolet light. The pigment is produced by a biosynthetic pathway, the genes of which are dispersed in the genome. The backbone molecule of the pigment is a polyketide synthesized by a polyketide synthase encoded by the wA gene. If wA is not functional, the conidia are white. The polyketide is modified by a laccase encoded by the yA gene and inactivation of yA in an otherwise wild-type background results in yellow spores. Additional spore color mutations have been isolated and mapped to a locus genetically, but the genes that correspond to these loci have not been determined. Spore color markers have been useful historically, and they remain valuable in the molecular genetics era. One can determine if a transforming fragment has been successfully integrated at the wA or yA locus by simply looking at the color of transformant conidia. The genes of the potentially useful color loci chaA (chartreuse conidia) and fwA (fawn conidia) have not been identified previously. We chose a set of candidate genes for each locus by comparing the assembled genome with the genetic map. By systematically deleting these candidate genes, we identified a cytochrome P450 gene (AN10028) corresponding to chaA . Deletions of this gene result in chartreuse conidia and chartreuse mutations can be complemented in trans by a functional copy of this gene. With fwA , we found that the existing fawn mutation, fwA1 , is a deletion of 2241 base pairs that inactivates three genes. By deleting each of these genes, we determined that fwA is AN1088, an EthD domain protein. Deletion of AN1088 results in fawn conidia as expected. Neither deletion of chaA nor fwA restricts growth and both should be valuable target loci for transformations. Combinations of deletions have allowed us to investigate the epistasis relationships of wA , yA , chaA and fwA .

Functions of protein SUMOylation remain incompletely understood in different cell types. Via forward genetics, here we identified ubaBQ247*, a loss-of-function mutation in a SUMO activation enzyme UbaB in the filamentous fungus Aspergillus nidulans. The ubaBQ247*, ΔubaB, and ΔsumO mutants all produce abnormal chromatin bridges, indicating the importance of SUMOylation in the completion of chromosome segregation. The bridges are enclosed by nuclear membrane containing peripheral nuclear pore complex proteins that normally get dispersed during mitosis, and the bridges are also surrounded by cytoplasmic microtubules typical of interphase cells. Time-lapse sequences further indicate that most bridges persist through interphase prior to the next mitosis, and anaphase chromosome segregation can produce new bridges that persist into the next interphase. When the first mitosis happens at a higher temperature of 42°C, SUMOylation deficiency produces not only chromatin bridges but also many abnormally shaped single nuclei that fail to divide. UbaB-GFP localizes to interphase nuclei just like the previously studied SumO-GFP, but the nuclear signals disappear during mitosis when the nuclear pores are partially open, and the signals reappear after mitosis. The nuclear localization is consistent with many SUMO targets being nuclear proteins. Finally, although the budding yeast SUMOylation machinery interacts with LIS1, a protein critical for dynein activation, loss of SUMOylation does not cause any obvious defect in dynein-mediated transport of nuclei and early endosomes, indicating that SUMOylation is unnecessary for dynein activation in A. nidulans., Competing Interests: Conflicts of interest The authors declare no conflict of interest., (Published by Oxford University Press on behalf of The Genetics Society of America 2023.)

Production of secondary metabolites is controlled by a complicated regulatory network in eukaryotic cells. Several layers of regulators are involved in this process, ranging from pathway-specific regulation, to epigenetic control, to global regulation. Here, we discover that interaction of an RNA-binding protein CsdA with a regulator RsdA coordinates fungal secondary metabolism. Employing a genetic deletion approach and transcriptome analysis as well as metabolomics analysis, we reveal that CsdA and RsdA synergistically regulate fungal secondary metabolism comprehensively. Mechanistically, comprehensive genetic and biochemical studies prove that RsdA and CsdA co-localize in the nucleus and physically interact to achieve their functions. In particular, we demonstrate that CsdA mediates rsdA expression by binding specific motif "GUCGGUAU" of its pre-mRNA at a post-transcriptional level. We thus uncover a mechanism in which RNA-binding protein physically interacts with, and controls the expression level of, the RsdA to coordinate fungal secondary metabolism., (© 2023. The Author(s).)

Aspergillus fumigatus is a serious human pathogen causing life-threatening Aspergillosis in immunocompromised patients. Secondary metabolites (SMs) play an important role in pathogenesis, but the products of many SM biosynthetic gene clusters (BGCs) remain unknown. In this study, we have developed a heterologous expression platform in Aspergillus nidulans , using a newly created genetic dereplication strain, to express a previously unknown BGC from A. fumigatus and determine its products. The BGC produces sartorypyrones, and we have named it the spy BGC. Analysis of targeted gene deletions by HRESIMS, NMR, and microcrystal electron diffraction (MicroED) enabled us to identify 12 products from the spy BGC. Seven of the compounds have not been isolated previously. We also individually expressed the polyketide synthase (PKS) gene spyA and demonstrated that it produces the polyketide triacetic acid lactone (TAL), a potentially important biorenewable platform chemical. Our data have allowed us to propose a biosynthetic pathway for sartorypyrones and related natural products. This work highlights the potential of using the A. nidulans heterologous expression platform to uncover cryptic BGCs from A. fumigatus and other species, despite the complexity of their secondary metabolomes., Competing Interests: The authors declare no conflicts of interest., (This journal is © The Royal Society of Chemistry.)

Polystyrene (PS) is one of the most used yet infrequently recycled plastics. Although manufactured on the scale of 300 million tons per year globally, current approaches toward PS degradation are energy- and carbon-inefficient, slow, and/or limited in the value that they reclaim. We recently reported a scalable process to degrade post-consumer polyethylene-containing waste streams into carboxylic diacids. Engineered fungal strains then upgrade these diacids biosynthetically to synthesize pharmacologically active secondary metabolites. Herein, we apply a similar reaction to rapidly convert PS to benzoic acid in high yield. Engineered strains of the filamentous fungus Aspergillus nidulans then biosynthetically upgrade PS-derived crude benzoic acid to the structurally diverse secondary metabolites ergothioneine, pleuromutilin, and mutilin. Further, we expand the catalog of plastic-derived products to include spores of the industrially relevant biocontrol agent Aspergillus flavus Af36 from crude PS-derived benzoic acid.

Secondary metabolites (SMs) are biologically active small molecules, many of which are medically valuable. Fungal genomes contain vast numbers of SM biosynthetic gene clusters (BGCs) with unknown products, suggesting that huge numbers of valuable SMs remain to be discovered. It is challenging, however, to identify SM BGCs, among the millions present in fungi, that produce useful compounds. One solution is resistance gene-guided genome mining, which takes advantage of the fact that some BGCs contain a gene encoding a resistant version of the protein targeted by the compound produced by the BGC. The bioinformatic signature of such BGCs is that they contain an allele of an essential gene with no SM biosynthetic function, and there is a second allele elsewhere in the genome. We have developed a computer-assisted approach to resistance gene-guided genome mining that allows users to query large databases for BGCs that putatively make compounds that have targets of therapeutic interest. Working with the MycoCosm genome database, we have applied this approach to look for SM BGCs that target the proteasome β6 subunit, the target of the proteasome inhibitor fellutamide B, or HMG-CoA reductase, the target of cholesterol reducing therapeutics such as lovastatin. Our approach proved effective, finding known fellutamide and lovastatin BGCs as well as fellutamide- and lovastatin-related BGCs with variations in the SM genes that suggest they may produce structural variants of fellutamides and lovastatin. Gratifyingly, we also found BGCs that are not closely related to lovastatin BGCs but putatively produce novel HMG-CoA reductase inhibitors., One-Sentence Summary: A new computer-assisted approach to resistance gene-directed genome mining is reported along with its use to identify fungal biosynthetic gene clusters that putatively produce proteasome and HMG-CoA reductase inhibitors., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society of Industrial Microbiology and Biotechnology.)

Waste plastics represent major environmental and economic burdens due to their ubiquity, slow breakdown rates, and inadequacy of current recycling routes. Polyethylenes are particularly problematic, because they lack robust recycling approaches despite being the most abundant plastics in use today. We report a novel chemical and biological approach for the rapid conversion of polyethylenes into structurally complex and pharmacologically active compounds. We present conditions for aerobic, catalytic digestion of polyethylenes collected from post-consumer and oceanic waste streams, creating carboxylic diacids that can then be used as a carbon source by the fungus Aspergillus nidulans. As a proof of principle, we have engineered strains of A. nidulans to synthesize the fungal secondary metabolites asperbenzaldehyde, citreoviridin, and mutilin when grown on these digestion products. This hybrid approach considerably expands the range of products to which polyethylenes can be upcycled., (© 2022 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

For many years, two central, unanswered questions in cytoskeleton research were how microtubule assembly is nucleated and microtubule polarity established. The discoveries of γ-tubulin and γ-tubulin ring complexes were key advances that allowed these questions to be substantially answered. The discovery of γ-tubulin was the product of a genetic screen in Aspergillus nidulans for genes important for microtubule function. γ-Tubulin is a member of the tubulin superfamily of proteins, closely related to α- and β-tubulin but distinct from both. It is ubiquitous in eukaryotes, and in many organisms there are small families of γ-tubulin genes. γ-Tubulin and associated proteins form ring-like complexes that localize to microtubule-organizing centers (MTOCs) and play an important role in the nucleation of microtubule assembly from MTOCs and the establishment of microtubule polarity.

Genome sequencing of filamentous fungi has demonstrated that most secondary metabolite biosynthetic gene clusters (BGCs) are silent under standard laboratory conditions. In this work, we have established an in vitro CRISPR-Cas9 system in Aspergillus wentii . To activate otherwise silent BGCs, we deleted the negative transcriptional regulator mcrA . Deletion of mcrA ( mcrA Δ) resulted in differential production of 17 SMs in total when the strain was cultivated on potato dextrose media (PDA). Nine out of fifteen of these SMs were fully characterized, including emodin ( 1 ), physcion ( 2 ), sulochrin ( 3 ), physcion bianthrone ( 4 ), 14- O -demethylsulochrin ( 5 ), ( trans / cis )-emodin bianthrone ( 6 and 7 ), and ( trans / cis )-emodin physcion bianthrone ( 8 and 9 ). These compounds were all found to be produced by the same polyketide synthase (PKS) BGC. We then performed a secondary knockout targeting this PKS cluster in the mcrA Δ background. The metabolite profile of the dual-knockout strain revealed new metabolites that were not previously detected in the mcrA Δ parent strain. Two additional SMs were purified from the dual-knockout strain and were characterized as aspergillus acid B ( 16 ) and a structurally related but previously unidentified compound ( 17 ). For the first time, this work presents a facile genetic system capable of targeted gene editing in A. wentii. This work also illustrates the utility of performing a dual knockout to eliminate major metabolic products, enabling additional SM discovery.

The microtubule-associated protein tau promotes the stabilization of the axonal cytoskeleton in neurons. In several neurodegenerative diseases, such as Alzheimer's disease, tau has been found to dissociate from microtubules, leading to the formation of pathological aggregates that display an amyloid fibril-like structure. Recent structural studies have shown that the tau filaments isolated from different neurodegenerative disorders have structurally distinct fibril cores that are specific to the disease. These "strains" of tau fibrils appear to propagate between neurons in a prion-like fashion that maintains their initial template structure. In addition, the strains isolated from diseased tissue appear to have structures that are different from those made by the most commonly used in vitro modeling inducer molecule, heparin. The structural differences among strains in different diseases and in vitro -induced tau fibrils may contribute to recent failures in clinical trials of compounds designed to target tau pathology. This study identifies an isoquinoline compound (ANTC-15) isolated from the fungus Aspergillus nidulans that can both inhibit filaments induced by arachidonic acid (ARA) and disassemble preformed ARA fibrils. When compared to a tau aggregation inhibitor currently in clinical trials (LMTX, LMTM, or TRx0237), ANTC-15 and LMTX were found to have opposing inducer-specific activities against ARA and heparin in vitro -induced tau filaments. These findings may help explain the disappointing results in translating potent preclinical inhibitor candidates to successful clinical treatments.

RNA-binding protein Musashi-1 (MSI1) is a key regulator of several stem cell populations. MSI1 is involved in tumor proliferation and maintenance, and it regulates target mRNAs at the translational level. The known mRNA targets of MSI1 include Numb , APC , and P21 WAF-1 , key regulators of Notch/Wnt signaling and cell cycle progression, respectively. In this study, we aim to identify small molecule inhibitors of MSI1-mRNA interactions, which could block the growth of cancer cells with high levels of MSI1. Using a fluorescence polarization (FP) assay, we screened small molecules from several chemical libraries for those that disrupt the binding of MSI1 to its consensus RNA. One cluster of hit compounds is the derivatives of secondary metabolites from Aspergillus nidulans . One of the top hits, Aza-9, from this cluster was further validated by surface plasmon resonance and nuclear magnetic resonance spectroscopy, which demonstrated that Aza-9 binds directly to MSI1, and the binding is at the RNA binding pocket. We also show that Aza-9 binds to Musashi-2 (MSI2) as well. To test whether Aza-9 has anti-cancer potential, we used liposomes to facilitate Aza-9 cellular uptake. Aza-9-liposome inhibits proliferation, induces apoptosis and autophagy, and down-regulates Notch and Wnt signaling in colon cancer cell lines. In conclusion, we identified a series of potential lead compounds for inhibiting MSI1/2 function, while establishing a framework for identifying small molecule inhibitors of RNA binding proteins using FP-based screening methodology.

In order for eukaryotes to efficiently detect and respond to environmental stimuli, a myriad of protein signaling pathways are utilized. An example of highly conserved signaling pathways in eukaryotes are the mitogen-activated protein kinase (MAPK) pathways. In fungi, MAPK pathways have been shown to regulate a diverse array of biological processes, such as asexual and sexual development, stress responses and the production of secondary metabolites (SMs). In the model fungus Aspergillus nidulans , a MAPK pathway known as the pheromone module is utilized to regulate both development and SM production. This signaling cascade consists of the three kinases SteC, MkkB, and MpkB, as well as the SteD adaptor protein and the HamE scaffold. In this study, homologs of each of these proteins have been identified in the opportunistic human pathogen A. fumigatus. By performing epitope tagging and mass spectrometry experiments, we have shown that these proteins form a pentameric complex, similar to what is observed in A. nidulans. This complex has been shown to assemble in the cytoplasm and MpkB enters the nucleus, where it would presumably interact with various transcription factors. Pheromone module mutant strains exhibit drastic reductions in asexual sporulation, vegetative growth rate and production of SMs, such as gliotoxin. Mutants also display increased sensitivity to cell wall and oxidative stress agents. Overall, these data provide evidence of the existence of a conserved MAP kinase signaling pathway in Aspergillus species and suggest that this pathway is critical for the regulation of fungal development and secondary metabolism., (Copyright © 2020 Frawley, Stroe, Oakley, Heinekamp, Straßburger, Fleming, Brakhage and Bayram.)

Aspergillus nidulans is an opportunistic fungal pathogen in patients with immunodeficiency, and virulence of A. nidulans isolates has mainly been studied in the context of chronic granulomatous disease (CGD), with characterization of clinical isolates obtained from non-CGD patients remaining elusive. This study therefore carried out a detailed biological characterization of two A. nidulans clinical isolates (CIs), obtained from a patient with breast carcinoma and pneumonia and from a patient with cystic fibrosis that underwent lung transplantation, and compared them to the reference, nonclinical FGSC A4 strain. Both CIs presented increased growth in comparison to that of the reference strain in the presence of physiologically relevant carbon sources. Metabolomic analyses showed that the three strains are metabolically very different from each other in these carbon sources. Furthermore, the CIs were highly susceptible to cell wall-perturbing agents but not to other physiologically relevant stresses. Genome analyses identified several frameshift variants in genes encoding cell wall integrity (CWI) signaling components. Significant differences in CWI signaling were confirmed by Western blotting among the three strains. In vivo virulence studies using several different models revealed that strain MO80069 had significantly higher virulence in hosts with impaired neutrophil function than the other strains. In summary, this study presents detailed biological characterization of two A. nidulans sensu stricto clinical isolates. Just as in Aspergillus fumigatus , strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits. Further studies are required to fully characterize A. nidulans strain-specific virulence traits and pathogenicity. IMPORTANCE Immunocompromised patients are susceptible to infections with opportunistic filamentous fungi from the genus Aspergillus Although A. fumigatus is the main etiological agent of Aspergillus species-related infections, other species, such as A. nidulans , are prevalent in a condition-specific manner. A. nidulans is a predominant infective agent in patients suffering from chronic granulomatous disease (CGD). A. nidulans isolates have mainly been studied in the context of CGD although infection with A. nidulans also occurs in non-CGD patients. This study carried out a detailed biological characterization of two non-CGD A. nidulans clinical isolates and compared the results to those with a reference strain. Phenotypic, metabolomic, and genomic analyses highlight fundamental differences in carbon source utilization, stress responses, and maintenance of cell wall integrity among the strains. One clinical strain had increased virulence in models with impaired neutrophil function. Just as in A. fumigatus , strain heterogeneity exists in A. nidulans clinical strains that can define virulence traits., (Copyright © 2020 Bastos et al.)

Fungal secondary metabolites (SMs) include medically valuable compounds as well as compounds that are toxic, carcinogenic, and/or contributors to fungal pathogenesis. It is consequently important to understand the regulation of fungal secondary metabolism. McrA is a recently discovered transcription factor that negatively regulates fungal secondary metabolism. Deletion of mcrA ( mcrA Δ), the gene encoding McrA, results in upregulation of many SMs and alters the expression of more than 1000 genes. One gene strongly upregulated by the deletion of mcrA is llmG , a putative methyl transferase related to LaeA, a major regulator of secondary metabolism. We artificially upregulated llmG by replacing its promoter with strong constitutive promoters in strains carrying either wild-type mcrA or mcrA Δ. Upregulation of llmG on various media resulted in increased production of the important toxin sterigmatocystin and compounds from at least six major SM pathways. llmG is, thus, a master SM regulator. mcrA Δ generally resulted in greater upregulation of SMs than upregulation of llmG , indicating that the full effects of mcrA on secondary metabolism involve genes in addition to llmG . However, the combination of mcrA Δ and upregulation of llmG generally resulted in greater compound production than mcrA Δ alone (in one case more than 460 times greater than the control). This result indicates that deletion of mcrA and/or upregulation of llmG can likely be combined with other strategies for eliciting SM production to greater levels than can be obtained with any single strategy.

SUMOylation, covalent attachment of the small ubiquitin-like modifier protein SUMO to proteins, regulates protein interactions and activity and plays a crucial role in the regulation of many key cellular processes. Understanding the roles of SUMO in these processes ultimately requires identification of the proteins that are SUMOylated in the organism under study. The filamentous fungus Aspergillus nidulans serves as an excellent model for many aspects of fungal biology, and it would be of great value to determine the proteins that are SUMOylated in this organism (i.e. its SUMOylome). We have developed a new and effective approach for identifying SUMOylated proteins in this organism in which we lock proteins in their SUMOylated state, affinity purify SUMOylated proteins using the high affinity S-tag, and identify them using sensitive Orbitrap mass spectroscopy. This approach allows us to distinguish proteins that are SUMOylated from proteins that are binding partners of SUMOylated proteins or are bound non-covalently to SUMO. This approach has allowed us to identify 149 proteins that are SUMOylated in A. nidulans. Of these, 67 are predicted to be involved in transcription and particularly in the regulation of transcription, 21 are predicted to be involved in RNA processing and 16 are predicted to function in DNA replication or repair., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Eukaryotic striatin forms striatin-interacting phosphatase and kinase (STRIPAK) complexes that control many cellular processes including development, cellular transport, signal transduction, stem cell differentiation and cardiac functions. However, detailed knowledge of complex assembly and its roles in stress responses are currently poorly understood. Here, we discovered six striatin (StrA) interacting proteins (Sips), which form a heptameric complex in the filamentous fungus Aspergillus nidulans. The complex consists of the striatin scaffold StrA, the Mob3-type kinase coactivator SipA, the SIKE-like protein SipB, the STRIP1/2 homolog SipC, the SLMAP-related protein SipD and the catalytic and regulatory phosphatase 2A subunits SipE (PpgA), and SipF, respectively. Single and double deletions of the complex components result in loss of multicellular light-dependent fungal development, secondary metabolite production (e.g. mycotoxin Sterigmatocystin) and reduced stress responses. sipA (Mob3) deletion is epistatic to strA deletion by supressing all the defects caused by the lack of striatin. The STRIPAK complex, which is established during vegetative growth and maintained during the early hours of light and dark development, is mainly formed on the nuclear envelope in the presence of the scaffold StrA. The loss of the scaffold revealed three STRIPAK subcomplexes: (I) SipA only interacts with StrA, (II) SipB-SipD is found as a heterodimer, (III) SipC, SipE and SipF exist as a heterotrimeric complex. The STRIPAK complex is required for proper expression of the heterotrimeric VeA-VelB-LaeA complex which coordinates fungal development and secondary metabolism. Furthermore, the STRIPAK complex modulates two important MAPK pathways by promoting phosphorylation of MpkB and restricting nuclear shuttling of MpkC in the absence of stress conditions. SipB in A. nidulans is similar to human suppressor of IKK-ε(SIKE) protein which supresses antiviral responses in mammals, while velvet family proteins show strong similarity to mammalian proinflammatory NF-KB proteins. The presence of these proteins in A. nidulans further strengthens the hypothesis that mammals and fungi use similar proteins for their immune response and secondary metabolite production, respectively., Competing Interests: The authors have declared that no competing interests exist.

Fungi are a major source of valuable bioactive secondary metabolites (SMs). These compounds are synthesized by enzymes encoded by genes that are clustered in the genome. The vast majority of SM biosynthetic gene clusters are not expressed under normal growth conditions, and their products are unknown. Developing methods for activation of these silent gene clusters offers the potential for discovering many valuable new fungal SMs. While a number of useful approaches have been developed, they each have limitations, and additional tools are needed. One approach, upregulation of SM gene cluster-specific transcription factors that are associated with many SM gene clusters, has worked extremely well in some cases, but it has failed more often than it has succeeded. Taking advantage of transcription factor domain modularity, we developed a new approach. We fused the DNA-binding domain of a transcription factor associated with a silent SM gene cluster with the activation domain of a robust SM transcription factor, AfoA. Expression of this hybrid transcription factor activated transcription of the genes in the target cluster and production of the antibiotic (+)-asperlin. Deletion of cluster genes confirmed that the cluster is responsible for (+)-asperlin production, and we designate it the aln cluster. Separately, coinduction of expression of two aln cluster genes revealed the pathway intermediate (2 Z,4 Z,6 E)-octa-2,4,6-trienoic acid, a compound with photoprotectant properties. Our findings demonstrate the potential of our novel synthetic hybrid transcription factor strategy to discover the products of other silent fungal SM gene clusters.

We have identified the cyclin domain-containing proteins encoded by the genomes of 17 species of Aspergillus as well as 15 members of other genera of filamentous ascomycetes. Phylogenetic analyses reveal that the cyclins fall into three groups, as in other eukaryotic phyla, and, more significantly, that they are remarkably conserved in these fungi. All 32 species examined, for example, have three group I cyclins, cyclins that are particularly important because they regulate the cell cycle, and these are highly conserved. Within the group I cyclins there are three distinct clades, and each fungus has a single member of each clade. These findings are in marked contrast to the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe , and Candida albicans , which have more numerous group I cyclins. These results indicate that findings on cyclin function made with a model Aspergillus species, such as A. nidulans , are likely to apply to other Aspergilli and be informative for a broad range of filamentous ascomycetes. In this regard, we note that the functions of only one Aspergillus group I cyclin have been analysed (NimE Cyclin B of A. nidulans ). We have consequently carried out an analysis of the members of the other two clades using A. nidulans as our model. We have found that one of these cyclins, PucA, is essential, but deletion of PucA in a strain carrying a deletion of CdhA, an activator of the anaphase promoting complex/cyclosome (APC/C), is not lethal. These data, coupled with data from heterokaryon rescue experiments, indicate that PucA is an essential G 1 /S cyclin that is required for the inactivation of the APC/C-CdhA, which, in turn, allows the initiation of the S phase of the cell cycle. Our data also reveal that PucA has additional, non-essential, roles in the cell cycle in interphase. The A. nidulans member of the third clade (AN2137) has not previously been named or analyzed. We designate this gene clbA . ClbA localizes to kinetochores from mid G 2 until just prior to chromosomal condensation. Deletion of clbA does not affect viability. However, by using a regulatable promoter system new to Aspergillus , we have found that expression of a version of ClbA in which the destruction box sequences have been removed is lethal and causes a mitotic arrest and a high frequency of non-disjunction. Thus, although ClbA is not essential, its timely destruction is essential for viability, chromosomal disjunction, and successful completion of mitosis.

Technical advances in Aspergillus nidulans enable relatively easy deletion of genomic sequences, insertion of sequences into the genome and alteration of genomic sequences. To extend the power of this system we wished to create strains with several selectable markers in a common genetic background to facilitate multiple, sequential transformations. We have developed an approach, using the recycling of the pyrG selectable marker, that has allowed us to create new deletions of the biA, pabaA, choA, and lysB genes. We have deleted these genes in a strain that carries the commonly used pyrG89, riboB2, and pyroA4 mutations as well as a deletion of the sterigmatocystin gene cluster and a deletion of the nkuA gene, which greatly reduces heterologous integration of transforming sequences. The new deletions are fully, easily and cheaply supplementable. We have created a strain that carries seven selectable markers as well as strains that carry subsets of these markers. We have identified the homologous genes from Aspergillus terreus, cloned them and used them as selectable markers to transform our new strains. The newly created strains transform well and the new deletion alleles appear to be complemented fully by the A. terreus genes. In addition, we have used deep sequencing data to determine the sequence alterations of the venerable and frequently used pyrG89, riboB2 and pyroA4 alleles and we have reannotated the choA gene., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Spore pigmentation is very common in the fungal kingdom. The best studied pigment in fungi is melanin which coats the surface of single cell spores. What and how pigments function in a fungal species with multiple cell conidia is poorly understood. Here, we identified and deleted a polyketide synthase (PKS) gene PfmaE and showed that it is essential for multicellular conidial pigmentation and development in a plant endophytic fungus, Pestalotiopsis fici. To further characterize the melanin pathway, we utilized an advanced Aspergillus nidulans heterologous system for the expression of the PKS PfmaE and the Pfma gene cluster. By structural elucidation of the pathway metabolite scytalone in A. nidulans, we provided chemical evidence that the Pfma cluster synthesizes DHN melanin. Combining genetic deletion and combinatorial gene expression of Pfma cluster genes, we determined that the putative reductase PfmaG and the PKS are sufficient for the synthesis of scytalone. Feeding scytalone back to the P. fici ΔPfmaE mutant restored pigmentation and multicellular adherence of the conidia. These results cement a growing understanding that pigments are essential not simply for protection of spores from biotic and abiotic stresses but also for spore structural development., (© 2017 John Wiley & Sons Ltd.)

The RNA-binding protein Hu antigen R (HuR) binds to AU-rich elements (ARE) in the 3'-untranslated region (UTR) of target mRNAs. The HuR-ARE interactions stabilize many oncogenic mRNAs that play important roles in tumorigenesis. Thus, small molecules that interfere with the HuR-ARE interaction could potentially inhibit cancer cell growth and progression. Using a fluorescence polarization (FP) competition assay, we identified the compound azaphilone-9 (AZA-9) derived from the fungal natural product asperbenzaldehyde, binds to HuR and inhibits HuR-ARE interaction (IC50 ~1.2 μM). Results from surface plasmon resonance (SPR) verified the direct binding of AZA-9 to HuR. NMR methods mapped the RNA-binding interface of HuR and identified the involvement of critical RNA-binding residues in binding of AZA-9. Computational docking was then used to propose a likely binding site for AZA-9 in the RNA-binding cleft of HuR. Our results show that AZA-9 blocks key RNA-binding residues of HuR and disrupts HuR-RNA interactions in vitro. This knowledge is needed in developing more potent AZA-9 derivatives that could lead to new cancer therapy.

Fungal nonribosomal peptide synthetases (NRPSs) are megasynthetases that produce cyclic and acyclic peptides. In Aspergillus nidulans, the NRPS ivoA (AN10576) has been associated with the biosynthesis of grey-brown conidiophore pigments. Another gene, ivoB (AN0231), has been demonstrated to be an N-acetyl-6-hydroxytryptophan oxidase that putatively acts downstream of IvoA. A third gene, ivoC, has also been predicted to be involved in pigment biosynthesis based on publicly available genomic and transcriptomic information. In this paper, we report the replacement of the promoters of the ivoA, ivoB, and ivoC genes with the inducible promoter alcA in a single cotransformation. Co-overexpression of the three genes resulted in the production of a dark-brown pigment in hyphae. In addition, overexpression of each of the Ivo genes, ivoA-C, individually or in combination, allowed us to isolate intermediates and confirm the function of each gene. IvoA was found to be the first known NRPS to carry out the acetylation of the amino acid, tryptophan., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Fungal secondary metabolites (SMs) are extremely important in medicine and agriculture, but regulation of their biosynthesis is incompletely understood. We have developed a genetic screen in Aspergillus nidulans for negative regulators of fungal SM gene clusters and we have used this screen to isolate mutations that upregulate transcription of the non-ribosomal peptide synthetase gene required for nidulanin A biosynthesis. Several of these mutations are allelic and we have identified the mutant gene by genome sequencing. The gene, which we designate mcrA, is conserved but uncharacterized, and it encodes a putative transcription factor. Metabolite profiles of mcrA deletant, mcrA overexpressing, and parental strains reveal that mcrA regulates at least ten SM gene clusters. Deletion of mcrA stimulates SM production even in strains carrying a deletion of the SM regulator laeA, and deletion of mcrA homologs in Aspergillus terreus and Penicillum canescens alters the secondary metabolite profile of these organisms. Deleting mcrA in a genetic dereplication strain has allowed us to discover two novel compounds as well as an antibiotic not known to be produced by A. nidulans. Deletion of mcrA upregulates transcription of hundreds of genes including many that are involved in secondary metabolism, while downregulating a smaller number of genes., Competing Interests: The authors declare no conflict of interests., (© 2016 John Wiley & Sons Ltd.)

Fungal genome projects are revealing thousands of cryptic secondary metabolism (SM) biosynthetic gene clusters that encode pathways that potentially produce valuable compounds. Heterologous expression systems should allow these clusters to be expressed and their products obtained, but approaches are needed to identify the most valuable target clusters. The inp cluster of Aspergillus nidulans contains a gene, inpE, that encodes a proteasome subunit, leading us to hypothesize that the inp cluster produces a proteasome inhibitor and inpE confers resistance to this compound. Previous efforts to express this cluster have failed, but by sequentially replacing the promoters of the genes of the cluster with a regulatable promotor, we have expressed them successfully. Expression reveals that the product of the inp cluster is the proteasome inhibitor fellutamide B, and our data allow us to propose a biosynthetic pathway for the compound. By deleting inpE and activating expression of the inp cluster, we demonstrate that inpE is required for resistance to internally produced fellutamide B. These data provide experimental validation for the hypothesis that some fungal SM clusters contain genes that encode resistant forms of the enzymes targeted by the compound produced by the cluster.

Onychomycosis (fungal nail infections) is very common worldwide but is fortunately not often lethal. Several powerful drugs have been introduced into clinical practice in recent years, but these infections remain difficult to cure primarily due to the difficulty of penetration of drug to the site of the infection in therapeutic concentrations. The nature of the disease, the causative fungi, and the characteristics of the drugs employed to treat this condition are discussed in this review.

To reduce the secondary metabolite background in Aspergillus nidulans and minimize the rediscovery of compounds and pathway intermediates, we created a "genetic dereplication" strain in which we deleted eight of the most highly expressed secondary metabolite gene clusters (more than 244,000 base pairs deleted in total). This strain allowed us to discover a novel compound that we designate aspercryptin and to propose a biosynthetic pathway for the compound. Interestingly, aspercryptin is formed from compounds produced by two separate gene clusters, one of which makes the well-known product cichorine. This raises the exciting possibility that fungi use differential regulation of expression of secondary metabolite gene clusters to increase the diversity of metabolites they produce., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In secondary metabolite biosynthesis, core synthetic genes such as polyketide synthase genes usually encode proteins that generate various backbone precursors. These precursors are modified by other tailoring enzymes to yield a large variety of different secondary metabolites. The number of core synthesis genes in a given species correlates, therefore, with the number of types of secondary metabolites the organism can produce. In our study, heterologous expression of all the A. terreus NRPS-like genes showed that two NRPS-like proteins, encoded by atmelA and apvA , release the same natural product, aspulvinone E. In hyphae this compound is converted to aspulvinones whereas in conidia it is converted to melanin. The genes are expressed in different tissues and this spatial control is probably regulated by their own specific promoters. Comparative genomics indicates that atmelA and apvA might share a same ancestral gene and the gene apvA is located in a highly conserved region in Aspergillus species that contains genes coding for life-essential proteins. Our data reveal the first case in secondary metabolite biosynthesis in which the tissue specific production of a single compound directs it into two separate pathways, producing distinct compounds with different functions. Our data also reveal that a single trans -prenyltransferase, AbpB, prenylates two substrates, aspulvinones and butyrolactones, revealing that genes outside of contiguous secondary metabolism gene clusters can modify more than one compound thereby expanding metabolite diversity. Our study raises the possibility of incorporation of spatial, cell-type specificity in expression of secondary metabolites of biological interest and provides new insight into designing and reconstituting their biosynthetic pathways.

Tremendous progress has been made in understanding the functions of γ-tubulin and, in particular, its role in microtubule nucleation since the publication of its discovery in 1989. The structure of γ-tubulin has been determined, and the components of γ-tubulin complexes have been identified. Significant progress in understanding the structure of the γ-tubulin ring complex and its components has led to a persuasive model for how these complexes nucleate microtubule assembly. At the same time, data have accumulated that γ-tubulin has important but less well understood functions that are not simply a consequence of its function in microtubule nucleation. These include roles in the regulation of plus-end microtubule dynamics, gene regulation, and mitotic and cell cycle regulation. Finally, evidence is emerging that γ-tubulin mutations or alterations of γ-tubulin expression play an important role in certain types of cancer and in other diseases., (© 2015 Oakley et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)

Centrosome amplification is a hallmark of many types of cancer cells, and clustering of multiple centrosomes is critical for cancer cell survival and proliferation. Human kinesin-14 HSET/KFIC1 is essential for centrosome clustering, and its inhibition leads to the specific killing of cancer cells with extra centrosomes. Since kinesin-14 motor domains are conserved evolutionarily, we conceived a strategy of obtaining kinesin-14 inhibitors using Aspergillus nidulans, based on the previous result that loss of the kinesin-14 KlpA rescues the non-viability of the bimC4 kinesin-5 mutant at 42 °C. However, it was unclear whether alteration of BimC or any other non-KlpA protein would be a major factor reversing the lethality of the bimC4 mutant. Here we performed a genome-wide screen for bimC4 suppressors and obtained fifteen suppressor strains. None of the suppressor mutations maps to bimC. The vast majority of them contain mutations in the klpA gene, most of which are missense mutations affecting the C-terminal motor domain. Our study confirms that the bimC4 mutant is suitable for a cell-based screen for chemical inhibitors of kinesin-14. Since the selection is based on enhanced growth rather than diminished growth, cytotoxic compounds can be excluded., (Published by Elsevier Inc.)

The aggregation of the microtubule-associated protein tau is a seminal event in many neurodegenerative diseases, including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activities. We have previously screened Aspergillus nidulans secondary metabolites for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol. One aggregation inhibitor identified was asperbenzaldehyde, an intermediate in azaphilone biosynthesis. We therefore tested 11 azaphilone derivatives to determine their tau assembly inhibition properties in vitro. All compounds tested inhibited tau filament assembly to some extent, and four of the 11 compounds had the advantageous property of disassembling preformed tau aggregates in a dose-dependent fashion. The addition of these compounds to the tau aggregates reduced both the total length and number of tau polymers. The most potent compounds were tested in in vitro reactions to determine whether they interfere with tau's normal function of stabilizing microtubules (MTs). We found that they did not completely inhibit MT assembly in the presence of tau. These derivatives are very promising lead compounds for tau aggregation inhibitors and, more excitingly, for compounds that can disassemble pre-existing tau filaments. They also represent a new class of anti-tau aggregation compounds with a novel structural scaffold.

The spindle assembly checkpoint (SAC) plays a critical role in preventing mitotic errors by inhibiting anaphase until all kinetochores are correctly attached to spindle microtubules. In spite of the economic and medical importance of filamentous fungi, relatively little is known about the behavior of SAC proteins in these organisms. In our efforts to understand the role of γ-tubulin in cell cycle regulation, we have created functional fluorescent protein fusions of four SAC proteins in Aspergillus nidulans, the homologs of Mad2, Mps1, Bub1/BubR1 and Bub3. Time-lapse imaging reveals that SAC proteins are in distinct compartments of the cell until early mitosis when they co-localize at the spindle pole body. SAC activity is, thus, spatially regulated in A. nidulans. Likewise, Cdc20, an activator of the anaphase-promoting complex/cyclosome, is excluded from interphase nuclei, but enters nuclei at mitotic onset and accumulates to a higher level in mitotic nuclei than in the surrounding nucleoplasm before leaving in anaphase/telophase. The activity of this critical cell cycle regulatory complex is likely regulated by the location of Cdc20. Finally, the γ-tubulin mutation mipAD159 causes a nuclear-specific failure of nuclear localization of Mps1 and Bub1/R1 but not of Cdc20, Bub3 or Mad2., (© 2014 John Wiley & Sons Ltd.)

A facile genetic methodology in the filamentous fungus Aspergillus nidulans allowed exchange of the starter unit ACP transacylase (SAT) domain in the nonreduced polyketide synthase (NR-PKS) AfoE of the asperfuranone pathway with the SAT domains from 10 other NR-PKSs. The newly created hybrid with the NR-PKS AN3386 is able to accept a longer starter unit in place of the native substrate to create a novel aromatic polyketide in vivo.

Fungi are prolific producers of secondary metabolites (SMs) that show a variety of biological activities. Recent advances in genome sequencing have shown that fungal genomes harbor far more SM gene clusters than are expressed under conventional laboratory conditions. Activation of these "silent" gene clusters is a major challenge, and many approaches have been taken to attempt to activate them and, thus, unlock the vast treasure chest of fungal SMs. This review will cover recent advances in genome mining of SMs in Aspergillus nidulans. We will also discuss current updates in gene annotation of A. nidulans and recent developments in A. nidulans as a molecular genetic system, both of which are essential for rapid and efficient experimental verification of SM gene clusters on a genome-wide scale. Finally, we will describe advances in the use of A. nidulans as a heterologous expression system to aid in the analysis of SM gene clusters from other fungal species that do not have an established molecular genetic system.

The aggregation of the microtubule-associated protein tau is a significant event in many neurodegenerative diseases including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activity. We have screened Aspergillus nidulans secondary metabolites containing aromatic ring structures for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol and the previously identified aggregation inhibitor emodin as a positive control. While several compounds showed some activity, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde were potent aggregation inhibitors as determined by both a filter trap assay and electron microscopy. In this study, these three compounds were stronger inhibitors than emodin, which has been shown in a prior study to inhibit the heparin induction of tau aggregation with an IC50 of 1-5 µM. Additionally, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde reduced, but did not block, tau stabilization of microtubules. 2,ω-Dihydroxyemodin and asperthecin have similar structures to previously identified tau aggregation inhibitors, while asperbenzaldehyde represents a new class of compounds with tau aggregation inhibitor activity. Asperbenzaldehyde can be readily modified into compounds with strong lipoxygenase inhibitor activity, suggesting that compounds derived from asperbenzaldehyde could have dual activity. Together, our data demonstrates the potential of 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde as lead compounds for further development as therapeutics to inhibit tau aggregation in Alzheimer's disease and neurodegenerative tauopathies., (Georg Thieme Verlag KG Stuttgart · New York.)

Aspernidine A is a prenylated isoindolinone alkaloid isolated from the model fungus Aspergillus nidulans. A genome-wide kinase knockout library of A. nidulans was examined, and it was found that a mitogen-activated protein kinase gene, mpkA, deletion strain produces aspernidine A. Targeted gene deletions were performed in the kinase deletion background to identify the gene cluster for aspernidine A biosynthesis. Intermediates were isolated from mutant strains which provided information about the aspernidine A biosynthesis pathway.

Fungal secondary metabolites (SMs) are an important source of medically valuable compounds. Genome projects have revealed that fungi have many SM biosynthetic gene clusters that are not normally expressed. To access these potentially valuable, cryptic clusters, we have developed a heterologous expression system in Aspergillus nidulans . We have developed an efficient system for amplifying genes from a target fungus, placing them under control of a regulatable promoter, transferring them into A. nidulans , and expressing them. We have validated this system by expressing nonreducing polyketide synthases of Aspergillus terreus and additional genes required for compound production and release. We have obtained compound production and release from six of these nonreducing polyketide synthases and have identified the products. To demonstrate that the procedure allows transfer and expression of entire secondary metabolite biosynthetic pathways, we have expressed all the genes of a silent A. terreus cluster and demonstrate that it produces asperfuranone. Further, by expressing the genes of this pathway in various combinations, we have clarified the asperfuranone biosynthetic pathway. We have also developed procedures for deleting entire A. nidulans SM clusters. This allows us to remove clusters that might interfere with analyses of heterologously expressed genes and to eliminate unwanted toxins.

We reannotated the A. niger NR-PKS gene, e_gw1_19.204, and its downstream R domain gene, est_GWPlus_C_190476, as a single gene which we named dtbA. Heterologous expression of dtbA in A. nidulans demonstrated that DtbA protein produces two polyketides, 2,4-dihydroxy-3,5,6-trimethylbenzaldehyde (1) and 6-ethyl-2,4-dihydroxy-3,5-dimethylbenzaldehyde (2). Generation of DtbAΔR+TE chimeric PKSs by swapping the DtbA R domain with the AusA (austinol biosynthesis) or ANID_06448 TE domain enabled the production of two metabolites with carboxylic acids replacing the corresponding aldehydes.

Systemic fungal infections of humans and economically important animals are increasingly common throughout the world. These infections are severe and often hard to treat with existing safe, oral medications. Thus there has been increasing research on alternatives resulting in study of natural and synthetic inhibitors of 1,3-β-Glucan synthase (GS) and chitin synthase (CS)-enzymes important in the biosynthesis of fungal cell walls that are not utilized in human biochemistry. Some such agents have recently been introduced into parenteral clinical use. There is hope that safe agents of this type with oral activity may yet emerge. This active area of research and its historic context with alternative agents is reviewed herein.