Six new nor-terpene endoperoxides, diacarnusins A-F (1-6), along with five known ones (7-11), were isolated from the extract of the Red Sea sponge Diacarnus erythraeanus, which presented cytotoxic activity against the colon carcinoma cell line. The structure elucidation was performed by analysis of mass spectrometric and 1D and 2D NMR data. The relative and absolute configurations of the stereocenters of the compounds were deduced based on the Capon and Macleod empirical rule and comparison of the sign of their specific rotations with those of endoperoxides with proven absolute configurations. The cytotoxicity of the pure compounds was evaluated against a panel of cancer cell lines. The structure elucidation and biological activity of the compounds are discussed.

Fusarium wilt of banana, caused by Fusarium oxysporum f. sp. cubense race TR4 (Foc), is one of the most destructive diseases threatening global banana production, particularly the Cavendish cultivar. Conventional control strategies, including chemical treatments and quarantine, remain largely ineffective and unsustainable, underscoring the urgent need for alternative approaches. Biological control using rhizosphere-associated microorganisms offers a promising and environmentally friendly strategy. In this study, we isolated 436 bacterial strains from the rhizosphere of healthy banana plants and screened them for antifungal activity against Foc. Out of the screened isolates, 93 exhibited significant in-vitro inhibitions, and 64 of these were subsequently evaluated in greenhouse assays. We found that 22 strains reduced Fusarium wilt severity by 45 to 85% compared to untreated controls. Among them, two isolates, DDC20 and DDC\_NEW2, consistently demonstrated strong biocontrol activity. In addition, cell-free culture media (CFCM) and crude extracts inhibited spore germination in fluorescence-based assays, indicating the involvement of secreted antifungal metabolites. Microscopy and confocal observations of GFP tagged Foc revealed hyphal abnormalities in the presence of bacterial treatments, including swelling, irregular branching, and distortion, accompanied by excessive sporulation characterized by abundant microconidia, macroconidia, and chlamydospores. Whole-genome sequencing and comparative analyses placed both isolates within the genus Bacillus. Genome mining using antiSMASH identified multiple biosynthetic gene clusters encoding known antifungal compounds such as surfactin, fengycin, bacillibactin, and difficidin, as well as putative novel clusters. LCMS confirmed the presence of surfactin and fengycin in bacterial extracts, supporting the genomic predictions. Collectively, these findings highlight the potential of DDC20 and DDC\_NEW2 (related to Bacillus spp.) from the banana rhizosphere as effective biocontrol agents against Foc TR4. This integrated approach, combining phenotypic assays, microscopy, and genome mining, provides a strong foundation for the development of sustainable strategies to manage Fusarium wilt in banana cultivation. ### Competing Interest Statement The authors have declared no competing interest.

Bacterial strains in the rhizosphere secrete volatile organic compounds (VOCs) that play critical roles in interand intra-kingdom signaling, influencing both microbe-microbe and microbe-plant interactions. In this study we evaluated the plant growth-promoting effects of VOCs emitted by Bacillus sp. NYG5 on Arabidopsis thaliana, Nicotiana tabacum, and Cucumis sativus, focusing on VOC-induced alterations in plant metabolic pathways, rhizosphere microbial communities, and soil chemical properties. NYG5 VOCs enhanced plant biomass across all tested species and induced significant shifts in rhizosphere microbial community composition, specifically increasing relative abundance of Gammaproteobacteria and reducing Deltaproteobacteria (Linear discriminant analysis Effect Size, p < 0.05). Soil analysis revealed a considerable reduction in humic substance concentrations following VOCs exposure, as detected by fluorescent spectral analysis. Using SPME-GC-MS, several novel VOCs were identified, some of which directly promoted plant growth. Transcriptomic analysis of N. tabacum exposed to NYG5 VOCs demonstrated activation of pathways related to phenylpropanoid biosynthesis, sugar metabolism, and hormone signal transduction. Within the phenylpropanoid biosynthesis pathway, a significant upregulation (p adj = 1.16e-14) of caffeic acid 3-O-methyltransferase was observed, a key enzyme leading to lignin and suberin monomer biosynthesis. These results highlight the complex mechanisms through which bacterial VOCs influence plant growth, including metabolic modulation, rhizosphere microbiome restructuring, and soil chemical changes. Collectively, this study highlights the pivotal role of bacterial VOCs in shaping plant-microbesoil interactions.

Sarcopoterium spinosum is a medicinal plant, presenting glucose-lowering properties. The study aimed to identify the active components and their mechanisms of action. Bioguided fractionation was utilized to isolate the active molecules, followed by NMR and HRESI MS for their identification and structural elucidation. Binding to the insulin receptor (IR) and activation of the receptor were measured in vitro. Glucose-lowering effects were validated in vivo. A novel procyanidin trimer, named sarcocyanidin A (1, catechin-(4α-8)-epicatechin-(4β-8)-epicatechin), was identified. Sarcocyanidin A (1) activated insulin signaling in CHO-IR and L6 myotubes, while the IR inhibitor abolished this effect. IR autofluorescence and cell-based thermal shift assays indicate a direct interaction of sarcocyanidin A (1) with IR. Sarcocyanidin A (1) also activated insulin signaling and reduced blood glucose in mice. Sarcocyanidin A, a novel procyanidin trimer, mediates at least part of the antidiabetic properties of SSE, through activation of IR.

Echinocandins are important antifungal drugs that inhibit the activity of the membrane-bound glucan synthase complex, which is responsible for the synthesis of the fungal cell wall β-(1,3)-glucan. Echinocandin resistance, linked to mutations in Fks, the catalytic subunit of the glucan synthase complex, is on the rise, particularly in Candida species, the most common human fungal pathogens. In this study, we used molecular docking experiments between echinocandins and the recently reported structure of Fks to propose a model in which these drugs form a ternary complex with the enzyme and membrane lipids. We then used site-selective reductive dehydration of alcohols to generate dehydroxylated echinocandin derivatives, which we evaluated against a panel of Candida strains constructed by introducing resistance-conferring mutations. We found that removing the hemiaminal alcohol that drives alterations in the three-dimensional structures of the echinocandin reduced their efficacy. Conversely, eliminating the benzylic alcohol of echinocandins enhanced their efficacy by up to two orders of magnitude, depending on the resistance-conferring mutation. Our findings provide valuable insights into how site-selective modifications of echinocandins can be used to combat resistance to these clinically important antifungal drugs.

Amphotericin B, nystatin, and natamycin are the only polyene antifungals in clinical use and remain last-resort therapies for severe fungal infections. These agents disrupt fungal membranes through ergosterol binding, but how structural differences between the three polyenes influence subcellular distribution, and whether such distribution is linked to resistance, remains uncharacterized. To investigate these relationships, we developed fluorescent probes from each clinically used polyene by conjugating a common fluorophore to a conserved functional group, preserving the amphoteric nature and ergosterol-dependent antifungal activity of the parent drug while enabling live-cell imaging. This unified design allowed direct comparison of localization and trafficking across Candida species, including sterol biosynthesis mutants with a high level of polyene resistance. The probes revealed structure-specific distribution patterns, with resistant strains showing enhanced accumulation within the vacuole lumen for all three polyenes. These findings suggest that vacuolar sequestration contributes to polyene resistance and may represent an unrecognized aspect of the fungal stress response. More broadly, this work provides the first direct visual evidence that polyene structure dictates subcellular distribution patterns in fungal cells.

Marine sponges are well-known for hosting rich microbial communities. Sponges are the most prolific source of marine bioactive compounds, which are frequently synthesized by their associated microbiota. Calyx nicaeensis is an endemic Mediterranean sponge with scarce information regarding its (bioactive) secondary metabolites. East Mediterranean specimens of mesophotic C. nicaeensis have never been studied. Moreover, no research has inspected its associated bacteria. Thus, we studied the sponge’s bacterial diversity and examined bacterial interspecific interactions in search of a promising antibacterial candidate. Such novel antimicrobial agents are needed since extensive antibiotic use leads to bacterial drug resistance. Bacteria cultivation yielded 90 operational taxonomic units (OTUs). A competition assay enabled the testing of interspecific interactions between the cultured OTUs. The highest-ranked antagonistic bacterium, identified as Paenisporosarcina indica (previously never found in marine or cold habitats), was mass cultured, extracted, and separated using size exclusion and reversed-phase chromatographic methods, guided by antibacterial activity. A pure compound was isolated and identified as 3-oxy-anteiso-C15-fatty acid-lichenysin. Five additional active compounds await final cleaning; however, they are lichenysins and surfactins. These are the first antibacterial compounds identified from either the C. nicaeensis sponge or P. indica bacterium. It also revealed that the genus Bacillus is not an exclusive producer of lichenysin and surfactin.

Background Bacterial strains in the rhizosphere may secrete an array of volatile organic compounds (VOCs), many of which are vital for inter- and intra-kingdom signaling, modulating microbe-microbe and microbe-plant interactions. In this study, we tested the plant growth promoting effect of VOCs emitted by Bacillus sp. NYG5 on several plants, focusing on VOC-induced changes in plant metabolic pathways, soil chemical properties and rhizo-microbiome. Results NYG5 emitted VOCs were found to promote growth of Arabidopsis thaliana, Nicotiana tabacum and Cucumis sativus, while also altering the community composition of the rhizosphere microbiome. Furthermore, fluorescent spectral analysis of the soil showed a significant decrease in concentration of humic substances following exposure to NYG5-emitted VOCs. Utilizing SPME-GC-MS analysis, several novel VOCs were identified, some of which were shown to promote plant growth. Transcriptomic analysis of N. tabacum plants exposed to NYG5-emitted VOCs revealed the induction of several plant growth related pathways such as phenylpropanoid biosynthesis, metabolism of sugars, and signal transduction of plant hormones. Conclusions Exposure to NYG5-emitted VOCs significantly impacted several plant growth related pathways, increasing plant biomass. Additionally, these VOCs altered the community composition of the rhizosphere microbiome, and decreased the concentration of humic substances in the soil. These findings may suggest that bacterial VOCs significantly impact plant growth via various mechanisms.

Echinocandins are a class of antifungal drugs that inhibit the activity of the β-(1,3)-glucan synthase complex, which synthesizes fungal cell wall β-(1,3)-glucan. Echinocandin resistance is linked to mutations in the FKS gene, which encodes the catalytic subunit of the glucan synthase complex. We present a molecular-docking-based model that provides insight into how echinocandins interact with the target Fks protein: echinocandins form a ternary complex with both Fks and membrane lipids. We used reductive dehydration of alcohols to generate dehydroxylated echinocandin derivatives and evaluated their potency against a panel of Candida pathogens constructed by introducing resistance-conferring mutations in the FKS gene. We found that removing the hemiaminal alcohol, which drives significant conformational alterations in the modified echinocandins, reduced their efficacy. Conversely, eliminating the benzylic alcohol of echinocandins enhanced potency by up to two orders of magnitude, in a manner dependent upon the resistance-conferring mutation. Strains that have developed resistance to either rezafungin, the most recently clinically approved echinocandin, or its dehydroxylated derivative RZF-1, exhibit high resistance to rezafungin while demonstrating moderate resistance to RZF-1. These findings provide valuable insight for combating echinocandin resistance through chemical modifications.

Volatile organic compounds (VOCs) produced by bacteria play an important, yet relatively unexplored role in interactions between plants and phytopathogens. In this study, the soil bacterium Bacillus halotolerans NYG5 was identified as a potent biocontrol agent against several phytopathogenic fungi (Macrophomina phaseolina, Rhizoctonia solani, Pythium aphanidermatum, and Sclerotinia sclerotiorum) through the production of VOCs. NYG5emitted VOCs also inhibited the growth of bacterial pathogens (Agrobacterium tumefaciens, Xanthomonas campestris, Clavibacter michiganensis, and Pseudomonas syringae). When cultured in various growth media, NYG5 produced a variety of VOCs. Five distinct VOCs (2-methylbutanoic acid, 5-methyl-2-hexanone, 2,3-hexanedione, 2-ethyl-1-hexanol, and 6-methyl-2-heptanone) were identified using headspace GC-MS. 2,3-Hexanedione exhibited potent lethal effects on the tested phytopathogens and nematicidal activity against Meloidogyne javanica at a concentration of 50 ppm. In addition, 0.05 ppm 2,3-hexanedione stimulated the expression of pathogenesis-related genes 1 and 2 in Arabidopsis thaliana. Interestingly, 2,3-hexanedione is used as a food additive at higher concentrations than those tested in this study. Hence, 2,3-hexanedione is a promising biologically active compound that might serve as a sustainable alternative to common chemical pesticides and an elicitor of plant defense.

BACKGROUND:Fungal phytopathogens are a significant threat to crops and food security, and there is a constant need to develop safe and effective compounds that antagonize them. In-planta assays are complex and tedious and are thus not suitable for initial high-throughput screening of new candidate antifungal compounds. We propose an in vitro screening pipeline that integrates five rapid quantitative and qualitative methods to estimate the efficacy and mode of action of prospective antifungal compounds. RESULTS:The pipeline was evaluated using five documented antifungal compounds (benomyl, catechol, cycloheximide, 2,4-diacetylphloroglucinol, and phenylacetic acid) that have different modes of action and efficacy, against the model soilborne fungal pathogen Fusarium oxysporum f. sp. radicis cucumerinum. We initially evaluated the five compounds' ability to inhibit fungal growth and metabolic activity using green fluorescent protein (GFP)-labeled F. oxysporum and PrestoBlue staining, respectively, in multiwell plate assays. We tested the compounds' inhibition of both conidial germination and hyphal elongation. We then employed FUN-1 and SYTO9/propidium iodide staining, coupled to confocal microscopy, to differentiate between fungal growth inhibition and death at the cellular level. Finally, using a reactive oxygen species (ROS)-detection assay, we were able to quantify ROS production in response to compound application. CONCLUSIONS:Collectively, the proposed pipeline provides a wide array of quantitative and qualitative data on the tested compounds that can help pinpoint promising novel compounds; these can then be evaluated more vigorously using in planta screening assays. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Aeruginosins are common metabolites of cyanobacteria. In the course of re-isolation of the known aeruginosins KT608A and KT608B for bioassay studies, we isolated three new sulfated aeruginosins, named aeruginosins KT688 (1), KT718 (2), and KT575 (3), from the extract of a Microcystis cell mass collected during the 2016 spring bloom event in Lake Kinneret, Israel. The structures of the new compounds were established on the basis of analyses of the 1D and 2D NMR, as well as HRESIMS data. Marfey’s method, coupled with HR ESI LCMS and chiral HPLC, was used to establish the absolute configuration of the amino acid and hydroxyphenyl lactic acid residues, respectively. Compounds 1–3 were tested for inhibition of the serine protease trypsin, and compounds 1 and 2 were found to exhibit IC50 values of 2.38 and 1.43 µM, respectively.

Supplementary Figure 2: The cytotoxicity of 1 and 2 was examined in NSCLC U-1810 and normal lung fibroblasts (WI-38) following 24 h and 48 h of continuous exposure using MTT assay. Cell viability is given as % of cell survival as compared to DMSO solvent treated cells. Data shown is the mean of three independent experiments {plus minus}SEM. The IC30, IC50 and IC70 concentrations are given in Supplementary Table S3.

Supplementary Table 2: NMR data of (3S)-14-methyldocos-4E-en-1-yn-3-ol (2).

Supplementary Table 1: NMR data of (3S)-icos-4E-en-1-yn-3-ol (1).

Supplementary Table 3: Compound 1 and compound 2-induced cytotoxicity in NSCLC U-1810 and normal diploid fibroblasts WI-38. Concentrations that induced 30 %, 50 % and 70 % (IC30, IC50 and 70) of cell viability reduction upon treatment with 1 or 2 for 24 h, 48 h or 72 h in NSCLC U-1810 and normal diploid fibroblasts WI-38 are given. The Area under the dose-response curve (AUC) is calculated for each treatment. The data presented is based on calculation from 3 independent biological replicates and the entire growth curves are shown in Supplementary Figure S2.

Supplementary Figure 1: Separation of Cribrochalina vasculum crude extract and isolation of compounds 1 and 2. (A) The crude extract (43 g) was separated by reversed phase flash chromatography using a gradient of solvents, from 100 % water through 100 % methanol and then to 100 % ethyl acetate. Fraction 10 was further separated twice on a size exclusion Sephadex LH-20 column using 1:1 mixture of chloroform:methanol as mobile phase. The semi-pure fractions from the second Sephadex LH-20 column (fractions 8-11) were combined and separated on a reversed phase HPLC column (semi-preparative YMC C-8) with a mixture of 40:51:9 acetonitrile:methanol:water as an isocratic mobile phase. Two pure substances, (3S)-icos-4E-en-1-yn-3-ol (1) (tR 41.8 min, 70.1 mg) and (3S)-14-methyldocos-4E-en-1-yn-3-ol (2) (tR 67.0 min, 109 mg) were obtained. (B) HPLC chromatogram of the semi-pure fractions 8-11 from the second Sephadex LH-20 column revealed two substances; (3S)-icos-4E-en-1-yn-3-ol (1) and (3S)-14-methyldocos-4E-en-1-yn-3-ol (2). (C) SMB MS fragmentation of compound 2 showed a loss of 14 mass units for each carbon except for C-14 where a loss of 28 mass units was observed, indicating that the branched methyl substituent is situated on C-14.

Supplementary Table 4: Compound 1 and compound 2 -induced cytotoxicity in tumor and normal cells of different origin. Concentrations that induced 30 %, 50 % and 70 % (IC30, IC50 and IC70) of cell viability reduction in tumor (U-1810, U-1285, H69, H82, A2780, SKOV-3) and normal cells (WI-38, cardiomyocytes, PBMC, BJ-5ta, BEAS-2B, RPE-1) treated for 72 h with compounds 1 or 2 are given. The Area under the dose-response curve (AUC) is calculated for each treatment. The data presented is based on calculation from 3 independent biological replicates and the entire growth curves are shown in Fig. 2C and Fig. 2D.

Supplementary Figure 3: (A) NSCLC U-1810 cells or diploid fibroblasts WI-38 were treated with DMSO, 1 or 2 (3 microM) for 48 h. Cisplatin (20 microM, 72 h) treated U-1810 cells were used as a positive control for induction of apoptotic morphology. Nuclear morphology of cells was examined after fixation in 4 % paraformaldehyde and staining with DAPI. Pictures showing nuclear morphology (magnification 60x, the scale bar equals 25 microm). Arrows show apoptotic morphology of the cell nuclei. (B) NSCLC U-1810 cells were treated with 3 microM concentration of compound 2 and cleavage of caspase-3, caspase-9 or PARP was examined after 24, 48 and 72 h. β actin was used to visualize equal loading.

Supplementary Materials and Methods and Supplementary Figure Legend