Toyonobu Usuki

Ecological interactions in the soil are often mediated by small molecules, which can later become valuable drugs. The cellular slime mould Dictyostelium discoideum is a soil microbe with a life cycle consisting of unicellular (amoeba) and multicellular phases (fruiting bodies). After Dictyostelium amoebae have consumed all available bacteria, they form stalked fruiting bodies to aid dispersal of the spores. The dying stalk cells repurpose a hybrid polyketide synthase to make abundant chlorinated metabolites, which persist in their fruiting bodies. The most abundant of the chlorinated metabolites, CDF-1, is a chlorinated dibenzofuran, which was shown to be an effective antimicrobial, being roughly as potent as ampicillin. Here, we identify CDF-2 and -3 by purification, followed by MS and NMR, after increasing their yields by using producer species and growth condition optimisation. Similar to CDF-1, CDF-2 and -3 are chlorinated dibenzofurans and exhibit more potent antibacterial activity against Gram-positive bacteria than ampicillin. We propose that the ecological function of CDF-2 and -3 is to protect the dormant spores from degradative bacteria.

Elastic lamellae are stratified extracellular structures essential for maintaining the integrity of large vessels. While numerous studies have elucidated the roles of individual molecules in elastic fiber formation, the mechanisms governing three-dimensional (3D) elastic fiber assembly in blood vessels remain incompletely understood. Advancing comprehensive understanding of these mechanisms requires overcoming the limitation of genetically modified animal models and conventional planar culture systems. Here, we present a 3D experimental vascular model (3D-VM) consisting of rat embryonic aortic smooth muscle cells (SMCs) that recapitulates multilayered SMCs and SMC-derived cross-linked elastic lamellae, constructed by a layer-by-layer technique utilizing fibronectin and gelatin. Electron microscopy confirmed the presence of stratified elastic lamellae, and mass spectrometry detected abundant desmosines and isodesmosines. The model exhibited an average burst pressure of 0.178 ± 0.042 MPa and withstood arterial pressures for at least 5 months after implantation in the adult rat aorta. Transcriptomic analysis revealed a gene expression profile in the 3D-VM that closely resembled that of native rat aortic tissues rather than planar-cultured SMCs. Gene ontology and pathway enrichment analyses identified significant positive correlations with genes associated with vascular development and extracellular matrix organization. Several elastic fiber-related genes were highly expressed at mRNA and protein levels in the 3D-VM compared with the adult aorta. Furthermore, fibulin-4 is a well-recognized elastic fiber component, and the 3D-VM generated with fibulin-4-deficient SMCs failed to form elastic fibers, highlighting the model's utility. These results suggest that the 3D-VM provides a platform for investigating the molecular mechanisms underlying 3D elastic fiber formation. STATEMENT OF SIGNIFICANCE: Elastic fibers confer tissues with distensibility and elastic recoil, allowing tissues to withstand repeated mechanical stress throughout life, particularly in dynamic organs such as arteries. Understanding the molecular mechanisms that govern elastic fiber formation is essential for developing therapeutic strategies for progressive diseases associated with elastic fiber dysfunction. To overcome the limitations of genetically modified animal models and conventional planar culture systems, which primarily elucidate the roles of individual molecules, we successfully established a three-dimensional vascular model composed of multilayered smooth muscle cells (SMCs) and SMC-derived cross-linked functional elastic lamellae. This model enables spatiotemporal analysis of elastic fiber formation and provides a platform for investigating the precise mechanisms that coordinate the interplay among multiple molecules.

BACKGROUND: Sophora Exigua Craib, also known as Phit-Sanat in Thai, belongs to the Fabaceae family. The root of S. exigua has been used in Kheaw-Hom, a Thai traditional remedy, for fever treatment. Bioactive compounds from S. exigua have been reported to exhibit health-promoting effects, including anticancer activity. However, their anti-leukemic properties have not yet been elucidated. METHODS: The study employed the MTT assay to evaluate the cytotoxic effects on leukemic cell lines (KG-1a and EoL-1) and PBMCs. Active compounds were purified using column chromatography and further characterized by NMR spectroscopy. Cell cycle distribution and apoptosis were assessed using appropriate kits and analyzed via flow cytometry. The expression of Wilms' tumor 1 (WT1) protein was examined by Western blot analysis. Proteomic analysis was conducted using online software to investigate gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. RESULTS: The ethyl acetate (EtOAc) crude fractional extract from S. exigua No. 010 (collected from Chaiyaphum province, Thailand) exhibited strong cytotoxicity in vitro toward both KG-1a and EoL-1 cells. Two active compounds, sophoraflavanone G (SG) and exiguaflavanone B (EGF-B), were isolated from EtOAc No. 010. EtOAc No. 010, SG, and EGF-B suppressed the proliferation of KG-1a and EoL-1 cells in a time- and dose-dependent manner by inducing G1 cell cycle arrest and apoptotic cell death. In this study, EtOAc No. 010, SG, and EGF-B were found to reduce WT1 expression in KG-1a and EoL-1 cells in a dose-dependent manner, with SG exhibiting greater activity than EGF-B. Additionally, KEGG pathway enrichment analysis of the differentially expressed proteins in KG-1a cells following SG treatment revealed significant enrichment in cell cycle regulation, apoptosis, and in the pathways associated with WT1protein expression, including AMPK, VEGF, and mTOR pathways. CONCLUSION: The SG isolated from S. exigua, exerts antiproliferative activity towards leukemic cells.

BACKGROUND: Desmosine and isodesmosine are crosslinking amino acids found in extracellular matrix protein elastin, which imparts elasticity to tissues such as those of the lungs and arteries. These compounds are promising biomarkers for diseases involving elastin degradation, such as chronic obstructive pulmonary disease. RESEARCH DESIGN AND METHOD: This study examined the correlation between isotope-dilution LC-MS/MS and a newly established ELISA for in vitro diagnosis using a variety of samples. RESULTS: Results of ELISA and LC-MS/MS analyses exhibited a high correlation coefficient (0.9941). However, whereas the LC-MS/MS measurements deviated approximately 2-fold from the theoretical values, the ELISA measurements ranged from 0.83 to 1.06 (avg 0.94) times the theoretical values. Precise measurement of the absorbance of synthetic desmosine revealed a molar extinction coefficient of 2403, which differed markedly from the previously reported value of 4900 in 1963. Using this value to recalculate the amount of added desmosine, the LC-MS/MS measurements were 0.68 to 0.99 (avg 0.87) times the theoretical values. CONCLUSION: Thus, the developed ELISA enables highly accurate determination of desmosine concentrations, comparable to LC-MS/MS, suggesting that ELISA is a potentially useful in vitro diagnostic tool.