H3K27me3 is vital for fungal development and secondary metabolite gene silencing, and substitutes for the loss of H3K9me3 in the plant pathogen Fusarium proliferatum.

Facultative heterochromatin marked by histone H3 lysine 27 trimethylation (H3K27me3) is an important regulatory layer involved in secondary metabolite (SM) gene silencing and crucial for fungal development in the genus Fusarium. While this histone mark is essential in some (e.g., the rice pathogen Fusarium fujikuroi), it appears dispensable in other fusaria. Here, we show that deletion of FpKMT6 is detrimental but not lethal in the plant pathogen Fusarium proliferatum, a member of the Fusarium fujikuroi species complex (FFSC). Loss of FpKmt6 results in aberrant growth, and expression of a large set of previously H3K27me3-silenced genes is accompanied by increased H3K27 acetylation (H3K27ac) and an altered H3K36me3 pattern. Next, H3K9me3 patterns are affected in Δfpkmt6, indicating crosstalk between both heterochromatic marks that became even more obvious in a strain deleted for FpKMT1 encoding the H3K9-specific histone methyltransferase. In Δfpkmt1, all H3K9me3 marks present in the wild-type strain are replaced by H3K27me3, a finding that may explain the subtle phenotype of the Δfpkmt1 strain which stands in marked contrast to other filamentous fungi. A large proportion of SM-encoding genes is allocated with H3K27me3 in the wild-type strain and loss of H3K27me3 results in elevated expression of 49% of them. Interestingly, genes involved in the biosynthesis of the phytohormones gibberellins (GA) are among the most upregulated genes in Δfpkmt6. Although several FFSC members harbor GA biosynthetic genes, its production is largely restricted to F. fujikuroi, possibly outlining the distinct lifestyles of these notorious plant pathogens. We show that H3K27me3 is involved in GA gene silencing in F. proliferatum and at least one additional FFSC member, and thus, may serve as a regulatory layer for gene silencing under non-favoring conditions.

A Sensitive LC-MS/MS Method for the Simultaneous Determination of Two Thia-Analogous Indirubin N-Glycosides and Indirubin-3'-Monoxime in Plasma and Cell Culture Medium.

Indirubin was identified as an active component of Danggui Longhui Wan, an herbal mixture used in traditional Chinese medicine, and showed anticancer activity in clinical trials in patients with chronic leukemia. Investigations on the mechanisms of antitumor action of indirubins have mainly focused on the indirubin derivative indirubin-3'-monoxime (I3M). Meanwhile, antiproliferative and cytotoxic properties on cancer cells have also been demonstrated for several synthetic indirubin N-glycosides. In the present study, we demonstrate cytotoxic activity of the thia-analogous indirubin N-glycosides KD87 (3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-glucopyranosyl)-oxindole) and KD85 (3-[3'-oxo-benzo[b]thiophen-2'-(Z)-ylidene]-1-(ß-d-mannopyranosyl)-oxindole) against melanoma and squamous cell carcinoma cells as well as lung cancer and glioblastoma cells. The advanced state of preclinical studies on the effects of indirubins conducted to date underscores the need for pharmacokinetic data from cellular, animal, and human studies for which reliable quantification is required. Therefore, a sensitive liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed and validated for the simultaneous measurement of KD87, KD85, and I3M in plasma and cell culture medium. Experimental conditions for sample preparation were optimized for human plasma protein precipitation and liquid-liquid extraction from plasma and cell culture medium. The methods were successfully validated in accordance with the U.S. Food and Drug Administration Bioanalytical Method Validation and evaluated for selectivity, sensitivity, matrix effect, recovery, carryover, calibration curve linearity, accuracy, precision, and stability. The applicability of the methods was demonstrated by the determination of KD87 in mouse plasma after prior intraperitoneal administration to mice.

Micro-scale screening of genetically modified Fusarium fujikuroi strain extends the apicidin family.

Apicidins are a class of naturally occurring cyclic tetrapeptides produced by few strains within the Fusarium genus. These secondary metabolites have gained significant attention due to their antiprotozoal activity through HDAC inhibition, thereby highlighting their potential for the treatment of malaria. Predominantly, apicidins have been isolated from Fusarium semitectum, offering a deep insight into the biosynthetic pathway responsible for their formation. A similar biosynthetic gene cluster has also been identified in the rice pathogenic fungus F. fujikuroi, leading the discovery of three additional apicidins through genetic manipulation. Routine mass spectrometric screening of these compound-producing strains revealed another metabolite structurally related to previously studied apicidins. By optimizing culture conditions and developing an effective isolation method, we obtained a highly pure substance, whose chemical structure was fully elucidated using NMR and HRMS fragmentation. Further studies were conducted to determine cytotoxicity, antimalarial activity, and HDAC inhibitory activity of this new secondary metabolite alongside the previously known apicidins. This work not only expands the apicidin class with a new member but also provides extensive insights and comparative analysis of apicidin-like substances produced by F. fujikuroi.