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Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the information network, whilst noncoding RNAs perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a cell. The first attempts to study the whole transcriptome began in the early 1990s, and technological advances since the late 1990s have made transcriptomics a widespread discipline. Transcriptomics has been defined by repeated technological innovations that transform the field. There are two key contemporary techniques in the field: microarrays, which quantify a set of predetermined sequences, and RNA sequencing (RNA-Seq), which uses high-throughput sequencing to capture all sequences. Measuring the expression of an organism's genes in different tissues, conditions, or time points gives information on how genes are regulated and reveals details of an organism's biology. It can also help to infer the functions of previously unannotated genes. Transcriptomic analysis has enabled the study of how gene expression changes in different organisms and has been instrumental in the understanding of human disease. An analysis of gene expression in its entirety allows detection of broad coordinated trends which cannot be discerned by more targeted assays.

Extracellular vesicles (EVs) represent a system for the coordinated secretion of a variety of molecular cargo including proteins, lipids, nucleic acids, and metabolites. They have an essential role in intercellular communication in multicellular organisms and have more recently been implicated in host-pathogen interactions. Study of the role for EVs in fungal biology has focused on pathogenic yeasts that are major pathogens in humans. In this study we have expanded the investigation of fungal EVs to plant pathogens, specifically the major cotton pathogen f. sp. . EVs isolated from f. sp. culture medium have a morphology and size distribution similar to EVs from yeasts such as and . A unique feature of the EVs from f. sp. is their purple color, which is predicted to arise from a napthoquinone pigment being packaged into the EVs. Proteomic analysis of f. sp. EVs revealed that they are enriched in proteins that function in synthesis of polyketides as well as proteases and proteins that function in basic cellular processes. Infiltration of f. sp. EVs into the leaves of cotton or plants led to a phytotoxic response. These observations lead to the hypothesis that f. sp. EVs are likely to play a crucial role in the infection process.

Antimicrobial peptides are a vital component of the innate immune system of all eukaryotic organisms and many of these peptides have potent antifungal activity. They have potential application in the control of fungal pathogens that are a serious threat to both human health and food security. Development of antifungal peptides as therapeutics requires an understanding of their mechanism of action on fungal cells. To date, most research on antimicrobial peptides has focused on their activity against bacteria. Several antimicrobial peptides specifically target fungal cells and are not active against bacteria. Others with broader specificity often have different mechanisms of action against bacteria and fungi. This review focuses on the mechanism of action of naturally occurring antifungal peptides from a diverse range of sources including plants, mammals, amphibians, insects, crabs, spiders, and fungi. While antimicrobial peptides were originally proposed to act via membrane permeabilization, the mechanism of antifungal activity for these peptides is generally more complex and often involves entry of the peptide into the cell.

Defensins are cationic antimicrobial peptides expressed throughout the plant and animal kingdoms as a first line of defense against pathogens. Membrane targeting and disruption is a crucial function of many defensins, however the precise mechanism remains unclear. Certain plant defensins form dimers that specifically bind the membrane phospholipids phosphatidic acid (PA) and phosphatidylinositol 4,5-bisphosphate, thereby triggering the assembly of defensin–lipid oligomers that permeabilize cell membranes. To understand this permeabilization mechanism, here we determine the crystal structure of the plant defensin NaD1 bound to PA. The structure reveals a 20-mer that adopts a concave sheet- or carpet-like topology where NaD1 dimers form one face and PA acyl chains form the other face of the sheet. Furthermore, we show that Arg39 is critical for PA binding, oligomerization and fungal cell killing. These findings identify a putative defensin–phospholipid membrane attack configuration that supports a longstanding proposed carpet mode of membrane disruption. Defensins are cationic antimicrobial peptides that permeabilize membranes of pathogens presumably via the assembly of defensin–lipid oligomers. Here authors provide evidence for this by solving the crystal structure of a plant defensin in an oligomeric state with phospholipids.

Transition metal ions are essential nutrients to all forms of life. Iron, copper, zinc, manganese, cobalt and nickel all have unique chemical and physical properties that make them attractive molecules for use in biological systems. Many of these same properties that allow these metals to provide essential biochemical activities and structural motifs to a multitude of proteins including enzymes and other cellular constituents also lead to a potential for cytotoxicity. Organisms have been required to evolve a number of systems for the efficient uptake, intracellular transport, protein loading and storage of metal ions to ensure that the needs of the cells can be met while minimizing the associated toxic effects. Disruptions in the cellular systems for handling transition metals are observed as a number of diseases ranging from hemochromatosis and anemias to neurodegenerative disorders including Alzheimer’s and Parkinson’s disease. The yeast Saccharomyces cerevisiae has proved useful as a model organism for the investigation of these processes and many of the genes and biological systems that function in yeast metal homeostasis are conserved throughout eukaryotes to humans. This review focuses on the biological roles of iron, copper, zinc, manganese, nickel and cobalt, the homeostatic mechanisms that function in S. cerevisiae and the human diseases in which these metals have been implicated.

Fusarium graminearum (Fgr) is a devastating filamentous fungal pathogen that causes diseases in cereals, while producing mycotoxins that are toxic for humans and animals, and render grains unusable. Low efficiency in managing Fgr poses a constant need for identifying novel control mechanisms. Evidence that fungal extracellular vesicles (EVs) from pathogenic yeast have a role in human disease led us to question whether this is also true for fungal plant pathogens. We separated EVs from Fgr and performed a proteomic analysis to determine if EVs carry proteins with potential roles in pathogenesis. We revealed that protein effectors, which are crucial for fungal virulence, were detected in EV preparations and some of them did not contain predicted secretion signals. Furthermore, a transcriptomic analysis of corn (Zea mays) plants infected by Fgr revealed that the genes of some of the effectors were highly expressed in vivo, suggesting that the Fgr EVs are a mechanism for the unconventional secretion of effectors and virulence factors. Our results expand the knowledge on fungal EVs in plant pathogenesis and cross-kingdom communication, and may contribute to the discovery of new antifungals.

Fusarium head blight, caused by Fusarium graminearum , is one of the most threatening fungal diseases of cereals worldwide. Current practices for control of F. graminearum are not always efficient, as epidemics still occur and there is low resistance in wheat varieties. Therefore, novel antifungal targets must be discovered by analyzing the molecular interaction between F. graminearum and its host. Fungal extracellular vesicles (EVs) are small membrane-bound compartments (30–1000 nm) that carry macromolecules and support fungal virulence, hence the disruption of EV production could lead to reduced fungal pathogenicity. However, EV study is limited by the lack of surface protein markers to aid in their characterization. Therefore, the aim of this report was to target a surface protein marker with an antibody, to unlock advanced EV characterization techniques. Using the list of potential EV markers for Candida albicans , we selected the tetraspanin-like Sur7 to perform immunogold microscopy, revealing that this protein is a surface marker of F. graminearum EVs. SUR7 is present on the surface of some but not all vesicles. EVs carrying SUR7 were larger than those without the marker, suggesting that there are subtypes of fungal EVs. The epitope recognized by the anti-Sur7 antibody is conserved in other Fusarium pathogens, making Sur7 a potential pan- Fusarium EV marker. Our results unlock techniques, such as immunoaffinity chromatography and antibody labeling, to track fungal EVs and understand their biogenesis, which may lead to the development of novel antifungals. Graphical abstract

Chitin is an essential polysaccharide of the fungal cell wall, critical for structural integrity, cell division and, in pathogenic fungi, virulence. As chitin is absent in both plant and mammalian systems, chitin synthases are considered attractive targets for the specific control of fungal pathogens. Yet despite decades of research, structural information on chitin synthases was lacking and inhibitors have failed to gain approval in the clinic. Current inhibitors are also ineffective against major agricultural pathogens such as Aspergillus and Fusarium species, largely due to the presence of multiple chitin synthase isoforms in filamentous fungi and the cell wall compensatory response induced under stress. However, recent cryo-electron microscopy structures of Class I chitin synthases from yeasts Saccharomyces cerevisiae and Candida albicans and an oomycete chitin synthase have provided unprecedented insights into the structural and mechanistic properties of these large, transmembrane proteins. These studies revealed conserved, domain-swapped homodimer architectures, distinct substrate binding and catalytic pockets, and sophisticated intrinsic regulatory mechanisms. With these breakthroughs, this review summarises our current understanding of fungal chitin biosynthesis, the challenges that remain to fully biochemically characterise these enzymes, and considers how the new structural insights may guide the development of broad-spectrum antifungals.

This was a phase I randomised comparator controlled clinical trial that assessed the pharmacokinetics (PK), and tolerability of IHL-675 A, a fixed dose combination (FDC) of cannabidiol (CBD) and hydroxychloroquine sulfate (HCQ), compared to the reference listed drugs Epidiolex (CBD) and Plaquenil (HCQ) in healthy volunteers (HVs). IHL-675 A is Incannex Healthcare Pty Ltd.’s proprietary product formulated using UniGel™ technology consisting of a solid, film coated HCQ tablet contained within a CBD-oil solution gel cap. Each IHL-675 A gel cap contains 75 mg of CBD and 100 mg HCQ. IHL-675 A is being developed for treatment of inflammatory conditions such as rheumatoid arthritis. This trial assessed the tolerability of IHL-675 A as well as pharmacokinetics of the active pharmaceutical ingredients CBD and HCQ as well as their major metabolites, compared to the reference listed drugs. The study included 3 treatment arms: IHL-675 A arm (150 mg CBD, 200 mg HCQ) and Plaquenil arm (200 mg HCQ) and Epidiolex (150 mg CBD) arm. Thirty-six participants were randomised into the 3 groups (12 per arm) and followed up for 4 weeks. Safety assessments including vital signs, electrocardiogram (ECG) parameters, and clinical laboratory parameters. Plasma concentrations of CBD, HCQ and their major metabolites, 7-OH-CBD, and 7-COOH-CBD, and desethylhydroxychloroquine (DHCQ), desethylchloroquine (DCQ), and bisdesethylhydroxy-chloroquine (BDCQ) were assessed at predefined timepoints across the monitoring period and PK parameters were determined and compared between treatments using noncompartmental methods and analysis of variance (ANOVA) of log-transformed values exposure PK parameters. All adverse events were coded using the current version of the Medical Dictionary for Regulatory Activities (MeDRA) by system organ class (SOC). IHL-675 A was generally well tolerated and had a similar adverse event profile compared to Epidiolex and Plaquenil. There were no SAEs reported in this study. Both CBD and HCQ were bioavailable when dosed in IHL-675 A. There were non-statistically significant differences between the pharmacokinetics of both CBD and HCQ when compared between IHL-675 A and Epidiolex or Plaquenil. There was approximately 50% increase in C max of CBD for IHL-675 A when compared with Epidiolex, and a slight increase of approximately 10% in AUC 0−last and AUC 0−inf . The 90% CI for the ratios of AUC 0−inf , AUC 0−last and C max of CBD all extended beyond the acceptance interval of 80% – 125%. The C max of HCQ for IHL-675 A was comparable to the reference product Plaquenil, with a geometric mean squares ratio of 96.5%. There was approximately 15% decrease in AUC 0−last . The 90% CI for the ratios of AUC 0−last and C max of HCQ extended beyond the acceptance interval of 80% – 125%. The ratio of AUC 0−inf was not reliable for comparison between treatments, since the t 1/2 was estimable for only 5 of 12 participants who received IHL-675 A. IHL-675 A was generally well tolerated when delivered as an oral fixed dose with no adverse events of concern or Serious Adverse Events (SAEs). Compared to the reference listed drugs for CBD (Epidiolex 150 mg) and HCQ (Plaquenil 200 mg), there was a slightly increased exposure to CBD and its metabolites for IHL-675 A, and slightly decreased exposure to HCQ. These results support the continued clinical development of IHL-675 A. Trial Registration : ACTRN12622000289718.

Plant defensins are a large family of small cationic proteins with diverse functions and mechanisms of action, most of which assert antifungal activity against a broad spectrum of fungi. The partial mechanism of action has been resolved for a small number of members of plant defensins, and studies have revealed that many act by more than one mechanism. The plant defensin Ppdef1 has a unique sequence and long loop 5 with fungicidal activity against a range of human fungal pathogens, but little is known about its mechanism of action. We screened the S. cerevisiae non-essential gene deletion library and identified the involvement of the mitochondria in the mechanism of action of Ppdef1. Further analysis revealed that the hyperpolarisation of the mitochondrial membrane potential (MMP) activates ROS production, vacuolar fusion and cell death and is an important step in the mechanism of action of Ppdef1, and it is likely that a similar mechanism acts in Trichophyton rubrum.