Metaphloem development in the Arabidopsis root tip.

The phloem transport network is a major evolutionary innovation that enabled plants to dominate terrestrial ecosystems. In the growth apices, the meristems, apical stem cells continuously produce early 'protophloem'. This is easily observed in Arabidopsis root meristems, in which the differentiation of individual protophloem sieve element precursors into interconnected conducting sieve tubes is laid out in a spatio-temporal gradient. The mature protophloem eventually collapses as the neighboring metaphloem takes over its function further distal from the stem cell niche. Compared with protophloem, metaphloem ontogenesis is poorly characterized, primarily because its visualization is challenging. Here, we describe the improved TetSee protocol to investigate metaphloem development in Arabidopsis root tips in combination with a set of molecular markers. We found that mature metaphloem sieve elements are only observed in the late post-meristematic root, although their specification is initiated as soon as protophloem sieve elements enucleate. Moreover, unlike protophloem sieve elements, metaphloem sieve elements only differentiate once they have fully elongated. Finally, our results suggest that metaphloem differentiation is not directly controlled by protophloem-derived cues but rather follows a distinct, robust developmental trajectory.

Synthesis of cytochalasan analogues with aryl substituents at position 10.

Cytochalasans are fungal metabolites that are known to inhibit actin polymerization. Despite their remarkable bioactivity, there are few studies on the structure-activity relationship (SAR) of the cytochalasan scaffold. The full potential of structural modifications remains largely unexplored. The substituent at position 10 of the cytochalasan scaffold is derived from an amino acid incorporated into the cytochalasan core, thus limiting the structural variability at this position in natural products. Additionally, modifications at this position have only been achieved through semisynthetic or mutasynthetic approaches using modified amino acids. This paper introduces a modular approach for late-stage modifications at position 10 of the cytochalasan scaffold. Iron-mediated cross-coupling reactions with corresponding Grignard reagents were used to introduce aryl or benzyl groups in position 10, resulting in the synthesis of six new cytochalasan analogues bearing non-natural aromatic residues. This methodology enables further exploration of modifications at this position and SAR studies among cytochalasan analogues.

Evolution-Based Discovery of Polyketide Acylated Valine from a Cytochalasin-Like Gene Cluster in Simplicillium lamelliciola HDN13430.

Utilizing a gene evolution-oriented approach for gene cluster mining, a cryptic cytochalasin-like gene cluster (sla) in Antarctic-derived Simplicillium lamelliciola HDN13430 was identified. Compared with the canonical cytochalasin biosynthetic gene clusters (BGCs), the sla gene cluster lacks the key α,ß-hydrolase gene. Heterologous expression of the sla gene cluster led to the discovery of a new compound, slamysin (1), characterized by an N-acylated amino acid structure and demonstrating weak anti-Bacillus cereus activity. These findings underscore the potential of genetic evolution in uncovering novel compounds and indicating specific adaptive evolution within specialized habitats.

Structure and Biosynthesis of Perochalasins A-C, Open-Chain Merocytochalasans Produced by the Marine-Derived Fungus Peroneutypa sp. M16.

Novel open-chain merocytochalasans, perochalasins A-C (1-3), containing an unusual N-O six-membered heterocyclic moiety, were isolated from cultures of the marine-derived Peroneutypa sp. M16 fungus, along with cytochalasin Z27 (4), cytochalasin Z28 (5), [12]-cytochalasin (6), and phenochalasin B (7). The structures of compounds 1-3 were established by analysis of the spectroscopic data. Full genome sequencing of Peroneutypa sp. M16 enabled the identification of a cytochalasan biosynthetic gene cluster and a proposal for the biosynthetic assembly of perochalasins. The proposal is supported by the nonenzymatic conversion of phenochalasin B (7) into 1-3, based on isotope-labeled hydroxylamine (15NH2OH and ND2OD) feeding studies in vivo and in vitro. In contrast to other merocytochalasans, these are the first cytochalasans confirmed to arise via nucleophilic addition and at a distinct location from the reactive macrocycle olefin, potentially expanding further the range of merocytochalasans to be discovered or engineered. Cytochalasin Z27 (4) exhibited antiplasmodial activities in the low micromolar range against the chloroquine-sensitive Plasmodium falciparum 3D7 strain as well as against resistant strains of the parasite (Dd2, TM90C6B, and 3D7r_MMV848).

Sucurchalasins A and B, Sulfur-Containing Heterodimers of a Cytochalasan and a Macrolide from the Endophytic Fungus Aspergillus spelaeus GDGJ-286.

Two sulfur-containing heterodimers of a cytochalasan and a macrolide, sucurchalasins A and B (1 and 2), and four known cytochalasan monomers (3-6), as well as four known macrolide derivatives (7-10), were obtained from the endophytic fungus Aspergillus spelaeus GDGJ-286. Sucurchalasins A and B (1 and 2) are the first cytochalasan heterodimers formed via a thioether bridge between cytochalasan and curvularin macrolide units. Their structures were elucidated by detailed analysis of NMR, LC-MS/MS, and X-ray crystallography. In bioassays, 1 and 2 exhibited cytotoxic effects on A2780 cells, with IC50 values of 3.9 and 8.3 µM, respectively. They also showed antibacterial activities against E. faecalis and B. subtilis with MIC values of 3.1 and 6.3 µg/mL, respectively.

Novel Metabolites from the Marine-Derived Fungus Peniophora sp. SCSIO41203 Show Promising In Vitro Antitumor Activity as Methuosis Inducers in PC-3 Cells.

Two new cytochalasin derivatives, peniotrinins A (1) and B (2), three new citrinin derivatives, peniotrinins C-E (4, 5, 7), and one new tetramic acid derivative, peniotrinin F (12), along with nine structurally related known compounds, were isolated from the solid culture of Peniophora sp. SCSIO41203. Their structures, including the absolute configurations of their stereogenic carbons, were fully elucidated based on spectroscopic analysis, quantum chemical calculations, and the calculated ECD. Interestingly, 1 is the first example of a rare 6/5/5/5/6/13 hexacyclic cytochalasin. We screened the above compounds for their anti-prostate cancer activity and found that compound 3 had a significant anti-prostate cancer cell proliferation effect, while compounds 1 and 2 showed weak activity at 10 µM. We then confirmed that compound 3 exerts its anti-prostate cancer effect by inducing methuosis through transmission electron microscopy and cellular immunostaining, which suggested that compound 3 might be first reported as a potential anti-prostate methuosis inducer.

The Cytochalasins and Polyketides from a Mangrove Endophytic Fungus Xylaria arbuscula QYF.

Twelve compounds, including four undescribed cytochalasins, xylariachalasins A-D (1-4), four undescribed polyketides (5-8), and four known cytochalasins (9-12), were isolated from the mangrove endophytic fungus Xylaria arbuscula QYF. Their structures and absolute configurations were established by extensive spectroscopic analyses (1D and 2D NMR, HRESIMS), electronic circular dichroism (ECD) calculations, 13C NMR calculation and DP4+ analysis, single-crystal X-ray diffraction, and the modified Mosher ester method. Compounds 1 and 2 are rare cytochalasin hydroperoxides. In bioactivity assays, Compound 2 exhibited moderate antimicrobial activities against Staphylococcus aureus and Candida albicans with MIC values of 12.5 µM for both Compound 10 exhibited significant cytotoxic activity against MDA-MB-435 with an IC50 value of 3.61 ± 1.60 µM.

Marcytoglobosins A and B, Cytochalasans From a Marine Sponge Associated Chaetomium globosum 162105 Fungus.

Two new cytochalasans, marcytoglobosins A (1) and B (2) were isolated from the marine sponge associated fungus Chaetomium globosum 162105, along with six known compounds (3-8). The complete structures of two new compounds were determined based on 1D/2D NMR and HR-MS spectroscopic analyses coupled with ECD calculations. All eight isolates were evaluated for their antibacterial activity. Among them, compounds 3-8 displayed antibacterial effects against Staphylococcus epidermidis, Bacillus thuringiensis, Pseudomonas syringae pv. Actinidiae, Vibrio alginolyticus, and Edwardsiella piscicida with minimum inhibitory concentration (MIC) values ranging from 10 to 25 µg/mL.

Sesquiterpenoids and Cytochalasins with Immunosuppressive Activity from Sonchus wightianus.

The plant species, Sonchus wightianus DC., was historically used in China for both medicinal and dietary uses. In present study, seven new guaiane sesquiterpenoids (1-7) and one cytochalasin (8), along with five known guaianes (9-13) and two known cytochalasins (14 and 15), were isolated from the whole plants of S. wightianus. These guaianes showed structural variations in the substituents at C-8 and/or C-15, and compounds 6 and 7 are two sesquiterpenoid glycoside derivatives. Their structures were determined by extensive analysis of spectroscopic, electronic circular dichroism, and X-ray diffraction data, and chemical method. Biological tests revealed that compounds 5 and 8 are potent and selective immunosuppressive reagents.

Diaporchalasins A-E, New Cytochalasins from the Endophytic Fungus Diaporthe sp. BMX12 Isolated from Aquilaria sinensis.

Five new cytochalasins, diaporchalasins A-E (1-5), together with 14 known congeners (6-19) were isolated from the endophytic fungus Diaporthe sp. BMX12, which was isolated from the branches of Aquilaria sinensis. The structures of the new compounds were elucidated by extensive spectroscopic analyses including high-resolution electron spray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR). Their absolute configurations were assigned by theoretical electronic circular dichroism (ECD) calculations. Compounds 11 and 12 featuring a keto carbonyl at C-21 displayed cytotoxicity toward K562, BEL-7402, SGC-7901, A549, and HeLa cell lines with IC50 values ranging from 4.4 to 47.4 µM.

Cytochalasans with Inhibitory Activity against NPC1L1 from the Endophytic Fungus Chaetomium nigricolor F5.

Mass spectrometry (MS)-based metabolic profiling of the endophytic fungus Chaetomium nigricolor F5 guided the isolation of five novel cytochalasans, chamisides B-F (1-5), and two known ones, chaetoconvosins C and D (6 and 7). Their structures including stereochemistry were unambiguously determined by MS, nuclear magnetic resonance, and single-crystal X-ray diffraction analyses. Compounds 1-3 share a new 5/6/5/5/7-fused pentacyclic skeleton in cytochalasans and are appropriately proposed to be the key biosynthetic precursors of co-isolated cytochalasans with a 6/6/5/7/5, 6/6/5/5/7, or 6/6/5 ring system. Remarkably, compound 5 with a relatively flexible side chain showed promising inhibition activity against the cholesterol transporter protein Niemann-Pick C1-like 1 (NPC1L1), expanding the function of cytochalasans.

Iron- and Reactive Oxygen Species-Dependent Ferroptotic Cell Death in Rice-Magnaporthe oryzae Interactions.

Hypersensitive response (HR) cell death is the most effective plant immune response restricting fungal pathogen invasion. Here, we report that incompatible rice (Oryza sativa) Magnaporthe oryzae interactions induce iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death in rice cells. Ferric ions and ROS (i.e., H2O2) accumulated in tissues undergoing HR cell death of rice leaf sheath tissues during avirulent M. oryzae infection. By contrast, iron did not accumulate in rice cells during virulent M. oryzae infection or treatment with the fungal elicitor chitin. Avirulent M. oryzae infection in ΔOs-nadp-me2-3 mutant rice did not trigger iron and ROS accumulation and suppressed HR cell death, suggesting that NADP-malic enzyme2 is required for ferroptotic cell death in rice. The small-molecule ferroptosis inhibitors deferoxamine, ferrostatin-1, and cytochalasin E and the NADPH oxidase inhibitor diphenyleneiodonium suppressed iron-dependent ROS accumulation and lipid peroxidation to completely attenuate HR cell death in rice sheaths during avirulent M. oryzae infection. By contrast, the small-molecule inducer erastin triggered iron-dependent ROS accumulation and glutathione depletion, which ultimately led to HR cell death in rice in response to virulent M. oryzae These combined results demonstrate that iron- and ROS-dependent signaling cascades are involved in the ferroptotic cell death pathway in rice to disrupt M. oryzae infection.

Cytochalasin Q exerts anti-melanoma effect by inhibiting creatine kinase B.

Due to the pivotal role of microfilament in cancer cells, targeting microfilaments with cytochalasins is considered a promising anticancer strategy. Here, we obtained cytochalasin Q (CQ) from Xylaria sp. DO1801, the endophytic fungi from the root of plant Damnacanthus officinarum, and discovered its anti-melanoma activity in vivo and in vitro attributing to microfilament depolymerization. Mechanistically, CQ directly bound to and inactivated creatine kinase B (CKB), an enzyme phosphorylating creatine to phosphocreatine (PCr) and regenerating ATP to cope with high energy demand, and then inhibited the creatine metabolism as well as cytosolic glycolysis in melanoma cells. Preloading PCr recovered ATP generation, reversed microfilament depolymerization and blunted anti-melanoma efficacy of CQ. Knockdown of CKB resulted in reduced ATP level, perturbed microfilament, inhibited proliferation and induced apoptosis, and manifested lower sensitivity to CQ. Further, we found that either CQ or CKB depletion suppressed the PI3K/AKT/FoxO1 pathway, whereas 740Y-P, a PI3K agonist, elevated protein expression of CKB suppressed by CQ. Taken together, our study highlights the significant anti-melanoma effect and proposes a PI3K/AKT/FoxO1/ CKB feedback circuit for the activity of CQ, opening new opportunities for current chemotherapy.

Cytotoxic Bromine- and Iodine-Containing Cytochalasins Produced by the Mangrove Endophytic Fungus Phomopsis sp. QYM-13 Using the OSMAC Approach.

Twelve new cytochalasins, phomopchalasins D-O (1-3, 5-12, and 14), including one brominated (2) and two iodinated cytochalasins (3 and 6), together with six known analogues (4, 13, and 15-18) were isolated from the mangrove-derived fungus Phomopsis sp. QYM-13 treated with 3% NaBr or 3% KI in potato liquid medium. Their structures and absolute configurations were established by extensive spectroscopic analysis (1D and 2D NMR, HRESIMS), electronic circular dichroism calculations, and a single-crystal X-ray diffraction experiment. Compounds 3 and 6 represent the first iodinated cytochalasins. Compounds 2, 15, 17, and 18 exhibited significant cytotoxicity against human cancer cell line MDA-MB-435 with IC50 values ranging from 0.2 to 8.2 µM.

TRAIL-sensitizing Cytochalasins from the Endophytic Fungus Phoma multirostrata.

Seven undescribed cytochalasins, multirostratins K - Q (2: -8: ), together with one known analogue, cytochalasin Z3 (1: ), were isolated from the culture of Phoma multirostrata XJ-2-1, an endophytic fungus obtained from the root of Parasenecio albus. Their structures with absolute configurations were determined by 1D and 2D NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), electronic circular dichroism (ECD), single-crystal X-ray crystallography, and chemical methods. The structure of ascochalasin was revised from Δ 13 to Δ 21 by detailed analysis of the NMR data and by comparison with the data for 7: . In a TRAIL (tumor necrosis factor related apoptosis inducing ligand)-resistance-overcoming experiment, co-treatment of 2: or 6: with TRAIL reduced the cell viability of A549 cells by 30.3% and 27.5% at 10 µM, respectively.

Cytochalasan Alkaloids as TRAIL Sensitizers from an Endophytic Fungus Chaetomium sp.

Two new cytochalasans with a rare 6/6/5/5/7 pentacyclic ring system, named chaetoconvosins C-D (1: -2: ), together with two known congeners (3: -4: ), were isolated from the fermentation of an endophytic fungus, Chaetomium sp. SG-01, harbored in the fibrous roots of Schisandra glaucescens Diels. Their structures including the absolute configuration were elucidated by extensive spectroscopic (HRESIMS, NMR, and ECD) and X-ray crystallographic analyses. The TRAIL-resistance-overcoming activity of 1: -4: in a TRAIL-resistant HT29 colorectal cancer cell line was evaluated, which revealed that co-treatment of 1: -4: at 50 µM with TRAIL (150 ng/mL) reduced the HT29 cell viability by 19.0%, 24.1%, 17.9%, and 15.5%, respectively, compared to treatment with 1: -4: alone.

Immunosuppressive Cytochalasins from the Mangrove Endophytic Fungus Phomopsis asparagi DHS-48.

Three new cytochalasins, phomoparagins A-C (1-3), along with five known analogs (4-8), were isolated from Phomopsis asparagi DHS-48, a mangrove-derived endophytic fungus. Their structures, including their absolute configurations, were elucidated using a combination of detailed HRESIMS, NMR, and ECD techniques. Notably, 1 possessed an unprecedented 5/6/5/8/5-fused pentacyclic skeleton. These compounds were tested for their inhibitory activity against concanavalin A (ConA)/lipopolysaccharide (LPS)-induced spleen lymphocyte proliferation and calcineurin (CN) enzyme. Several metabolites (2 and 4-6) exhibited fascinating inhibitory activities with a relatively low toxicity. Furthermore, 2 was demonstrated to inhibit ConA-stimulated activation of NFAT1 dephosphorylation and block NFAT1 translocation in vitro, subsequently inhibiting the transcription of interleukin-2 (IL-2). Our results provide evidence that 2 may, at least partially, suppress the activation of spleen lymphocytes via the CN/NFAT signaling pathway, highlighting that it could serve as an effective immunosuppressant that is noncytotoxic and natural.

Epigenetic Manipulation Induced Production of Immunosuppressive Chromones and Cytochalasins from the Mangrove Endophytic Fungus Phomopsis asparagi DHS-48.

A mangrove endophytic fungus Phomopsis asparagi DHS-48 was found to be particularly productive with regard to the accumulation of substantial new compounds in our previous study. In order to explore its potential to produce more unobserved secondary metabolites, epigenetic manipulation was used on this fungus to activate cryptic or silent genes by using the histone deacetylase (HDAC) inhibitor sodium butyrate and the DNA methyltransferase (DNMT) inhibitor 5-azacytidine (5-Aza). Based on colony growth, dry biomass, HPLC, and 1H NMR analyses, the fungal chemical diversity profile was significantly changed compared with the control. Two new compounds, named phaseolorin J (1) and phomoparagin D (5), along with three known chromones (2-4) and six known cytochalasins (6-11), were isolated from the culture treated with sodium butyrate. Their structures, including their absolute configurations, were elucidated using a combination of detailed HRESIMS, NMR, and ECD and 13C NMR calculations. The immunosuppressive and cytotoxic activities of all isolated compounds were evaluated. Compounds 1 and 8 moderately inhibited the proliferation of ConA (concanavalin A)-induced T and LPS (lipopolysaccharide)-induced B murine spleen lymphocytes. Compound 5 exhibited significant in vitro cytotoxicity against the tested human cancer cell lines Hela and HepG2, which was comparative to the positive control adriamycin and fluorouracil. Our finding demonstrated that epigenetic manipulation should be an efficient strategy for the induction of new metabolites from mangrove endophytic fungi.

Proteomic Analysis of the Sphincter in a Neurogenic Bladder Caused by T10 Spinal Cord Injury.

OBJECTIVE: This study aimed to conduct proteomic analysis of the sphincter in a neurogenic bladder caused by T10 spinal cord injury. The differentially expressed proteins (DEPs) of the sphincters (internal urethral sphincter) in the neurogenic bladders (NBs) of rats after complete transection of the T10 spinal cord segment were screened using tandem mass tag (TMT)-based quantitative labeling, and their biological information was analyzed. METHODS: Twelve adult Sprague Dawley rats out of 40 were randomly assigned to the blank group (n = 12), while the remaining 28 were placed in the T10 spinal cord injury model via modified Hassan Shaker spinal cord transection; 12 of these rats were then randomly selected as the model group. The rats in both groups underwent urodynamics detection and hematoxylin and eosin (H&E) staining. The proteins expressed in the bladder sphincter were detected using TMT-based quantitative proteomics. DEPs were defined as proteins with fold change >1.5 or <1/1.5, p < 0.05, and unique peptide ≥2. The DEPs were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using KOBAS 3.0., and gene ontology functional annotation analysis was performed using the Cytoscape 3.7.1. BiNGO plug-in. The protein-protein interaction network was then constructed using the interactive gene-retrieval tool STRING and Cytoscape software. RESULTS: The leak-point pressure and maximum cystometric volume in the model group were significantly higher than those in the blank group (p < 0.01), and H&E staining showed continuous interruption of the bladder sphincter fibers in the model group. A total of 250 DEPs were screened in the bladder sphincter, 83 of which were up-regulated and 167 of which were down-regulated. KEGG analysis of the DEPs was used to screen 15 pathways, including metabolic pathways, extracellular matrix (ECM)-receptor interaction, adhesion spots, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway, the cytochalasin signaling pathway, and the advanced glycation end-products (AGE)/receptor for AGEs (RAGE) signaling pathway in diabetic complications and vascular smooth muscle contraction. CONCLUSIONS: It is of great significance to explore the pathological mechanism of non-inhibitory contraction of the bladder sphincter caused by spinal cord injury above the T10 segment from the perspective of ECM-receptor interaction, focal adhesion-activated PI3K/Akt signaling pathway, and cell relaxation signaling pathways. Synaptic vesicle glycoprotein (Sv2A) involved in the release of neurotransmitters from synaptic vesicles, arrestin ß2 inhibitory proteins involved in α-adrenergic receptors and G-protein-coupled receptor internalization, and calmodulin and calmodulin binding protein involved in calcium-sensitive signaling pathways may be potential targets for developing new ways to treat bladder sphincter overactivity caused by T10 spinal cord injury.

New Cytochalasin Derivatives from Deep-Sea Cold Seep-Derived Endozoic Fungus Curvularia verruculosa CS-129.

Two new antimicrobial cytochalasin derivatives, 6ß,7ß-epoxydeoxaphomin C (1) and 12-hydroxydeoxaphomin C (2), a new natural occurring product 24-nor-cytochalasin B (3), together with two related known analogs (4-5) were isolated and identified from an endozoic fungus Curvularia verruculosa CS-129, isolated from the deep-sea squat lobster Shinkaia crosnieri which was collected in cold seep region of south China sea. The structures of new compounds were elucidated on the basis of detailed spectroscopic analysis and ECD calculation. The spectroscopic data of 24-nor-cytochalasin B (3) were reported for the first time. All compounds were tested for their antibacterial activities against human and aquatic pathogenic bacteria.