Fusarium solani PQF9 Isolated from Podocarpus pilgeri Growing in Vietnam as a New Producer of Paclitaxel.

Endophytic fungi are known as an alternative promising source of anticancer drug, paclitaxel, however fungi inhabiting in medicinal plant Podocarpus pilgeri and their paclitaxel production have not been reported to date. In the present study, a total of 15 culturable fungi classified into 5 genera, were successfully recovered from P. pilgeri collected in Vietnam. Screening fungal dichloromethane extracts for anticancer activity revealed that only PQF9 extract displayed potent inhibitory effects on A549 and MCF7 cancer cell lines with IC50 values of 33.9 ± 2.3 µg/mL and 43.5 ± 1.7 µg/mL, respectively. Through PCR-based molecular screening, the isolate PQF9 was found to possess 3 key genes involved in paclitaxel biosynthesis. Importantly, high-performance liquid chromatography quantification showed that fungal isolate PQF9 was able to produce 18.2 µg/L paclitaxel. The paclitaxel-producing fungus was identified as Fusarium solani PQF9 based on morphological and molecular phylogenetic analysis. Intensive investigations by chromatographic methods and spectroscopic analyses confirmed the presence of paclitaxel along with tyrosol and uracil. The pure paclitaxel had an IC50 value of 80.8 ± 9.4 and 67.9 ± 7.0 nM by using cell viability assay on A549 lung and MCF7 breast cancer cells. In addition, tyrosol exhibited strong antioxidant activity by scavenging 2, 2-diphenyl-picrylhydrazyl (DPPH) (IC50 5.1 ± 0.2 mM) and hydroxyl radical (IC50 3.6 ± 0.1 mM). In contrast, no biological activity was observed for uracil. Thus, the paclitaxel-producing fungus F. solani PQF9 could serve as a new material for large-scale production and deciphering paclitaxel biosynthesis. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01119-z.

Phenotypic and genomic characterization provide new insights into adaptation to environmental stressors and biotechnological relevance of mangrove Alcaligenes faecalis D334.

Alcaligenes faecalis D334 was determined in this study as a salt-tolerant bacterium isolated from mangrove sediment. In response to 6% (w/v) NaCl, strain D334 produced the highest ectoines of 14.14 wt%. To understand adaptive features to mangrove environment, strain D334 was sequenced using Pacific BioScience platform, resulting in a circular chromosome of 4.23 Mb. Of note, D334 genome harbored 81 salt-responsive genes, among which two membrane-associated genes ompc and eric were absent in 3 selected A. faecalis genomes. Apart from that, a complete pathway for ectoine and 5-hydroxyectoine synthesis was predicted. To resist 40 mM H2O2, 46 genetic determinants contributing to oxidative stress response were employed. Moreover, two operons involved in polyhydroxyalkanoate (PHA) production were identified in the D334 genome, resulting in maximum PHA content of 5.03 ± 0.04 wt% and PHA concentration of 0.13 ± 0.001 g/L. A large flagellar biosynthesis operon contributing to swimming motility was found to be conserved in D334 and 8 other A. faecalis genomes. These findings shed light for the first time on the high versatility of A. faecalis D334 genome to adapt to mangrove lifestyle and the possibility to develop D334 as an industrial platform for PHA and 5-hydroxyectoine production.

Extraction of cage-like sporopollenin exine capsules from dandelion pollen grains.

Pollen-based microcapsules such as hollow sporopollenin exine capsules (SECs) have emerged as excellent drug delivery and microencapsulation vehicles. To date, SECs have been extracted primarily from a wide range of natural pollen species possessing largely spherical geometries and uniform surface features. Nonetheless, exploring pollen species with more diverse architectural features could lead to new application possibilities. One promising class of candidates is dandelion pollen grains, which possess architecturally intricate, cage-like microstructures composed of robust sporopollenin biopolymers. Here, we report the successful extraction and macromolecular loading of dandelion SECs. Preservation of SEC morphology and successful removal of proteinaceous materials was evaluated using scanning electron microscopy (SEM), matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry, elemental CHN analysis, dynamic image particle analysis (DIPA) and confocal laser scanning microscopy (CLSM). Among the tested processing schemes, acidolysis using 85% (v/v) phosphoric acid refluxed at 70 °C for 5 hours yielded an optimal balance of intact particle yield, protein removal, and preservation of cage-like microstructure. For proof-of-concept loading, bovine serum albumin (BSA) was encapsulated within the dandelion SECs with high efficiency (32.23 ± 0.33%). Overall, our findings highlight how hollow microcapsules with diverse architectural features can be readily prepared and utilized from plant-based materials.

Safety assessment of myogenic stem cell transplantation and resulting tumor formation.

OBJECTIVES: To assess for stem cell migration to liver and lung after transplantation in injured rat anal sphincters. To evaluate histological findings of unanticipated ectopic foci of growth. METHODS: This is a prospective study involving 33 female virginal Sprague-Dawley rats. Anal sphincters were transected and repaired under sterile technique. Animals received injections of 5.0 × 10 myogenic stem cells (24 rats) or sham control (9 rats) and were killed on day 30. Liver and lung samples were obtained. Upon encountering abnormal foci of growth, further staining protocols were employed. Enzyme-linked immunosorbent assay studies evaluated stem cell media for in vitro growth factor secretion. RESULTS: No evidence of cell migration to liver or lung was found at the time of euthanasia in any study animal. Ectopic foci of growth were noted in 2 transplant rats. Further histological evaluations of these growths were consistent with benign tumors: no nuclear abnormalities and no evidence of proliferation at day 30. Enzyme-linked immunosorbent assay studies demonstrated positive secretion of vascular endothelial growth factor and insulin growth factor into the media of cultured rat myogenic stem cells. CONCLUSIONS: Whereas distant migration was not encountered in the liver or lung, 2 transplanted rats developed abnormal foci of growth, that is, tumors, from the external anal sphincter-raising further safety questions. Additional evaluation of these foci seemed benign. Possible explanations include cell trapping, stem cell overgrowth, and/or paracrine factors. The lack of cell migration supports that future investigation of safety parameters could focus locally.

Allogenic myoblast transplantation in the rat anal sphincter.

OBJECTIVES: : To determine the feasibility of injecting rat myoblasts into the intact anal sphincter as a potential treatment for anal incontinence, and to detect transferred myoblast survival and integration. STUDY DESIGN: : A pilot study using nonpregnant female Sprague Dawley rodents of 8 to 10 weeks of age. A biopsy of skeletal muscle was harvested from a study animal and recovered myoblasts were expanded in vitro over 10 days. Myoblasts were then tagged with a cytomegalovirus promoter to transduce green fluorescent protein (GFP) into the myoblasts. The cell aspirate was injected directly into the intact external anal sphincter using an electromyographic guidance. The animals received 1.5 or 4.5 × 10 cells of GFP-labeled myoblasts, dividing the dose between three injection sites. The remaining in vitro myoblasts were still viable 28 days post-harvest. Ten days after transplantation the anal sphincter complex was surgically extracted. RESULTS: : The presence of GFP-labeled myoblasts was confirmed within the external anal sphincter. CONCLUSIONS: : This demonstrates that myoblasts can be successfully extracted, cultivated in vitro, transplanted and will integrate into the host tissue.