A novel role of FoxO3a in the migration and invasion of trophoblast cells: from metabolic remodeling to transcriptional reprogramming.

BACKGROUND: The forkhead box O3a protein (FoxO3a) has been reported to be involved in the migration and invasion of trophoblast, but its underlying mechanisms unknown. In this study, we aim to explore the transcriptional and metabolic regulations of FoxO3a on the migration and invasion of early placental development. METHODS: Lentiviral vectors were used to knock down the expression of FoxO3a of the HTR8/SVneo cells. Western blot, matrigel invasion assay, wound healing assay, seahorse, gas-chromatography-mass spectrometry (GC-MS) based metabolomics, fluxomics, and RNA-seq transcriptomics were performed. RESULTS: We found that FoxO3a depletion restrained the migration and invasion of HTR8/SVneo cells. Metabolomics, fluxomics, and seahorse demonstrated that FoxO3a knockdown resulted in a switch from aerobic to anaerobic respiration and increased utilization of aromatic amino acids and long-chain fatty acids from extracellular nutrients. Furthermore, our RNA-seq also demonstrated that the expression of COX-2 and MMP9 decreased after FoxO3a knockdown, and these two genes were closely associated with the migration/invasion progress of trophoblast cells. CONCLUSIONS: Our results suggested novel biological roles of FoxO3a in early placental development. FoxO3a exerts an essential effect on trophoblast migration and invasion owing to the regulations of COX2, MMP9, aromatic amino acids, energy metabolism, and oxidative stress.

A New Species in Pseudophialophora From Wild Rice and Beneficial Potential.

Wild rice (Oryza granulata) is a natural resource pool containing abundant unknown endophytic fungi species. There are few reports on the endophytic fungi in wild rice. Here, one isolate recovered from wild rice roots was identified as a new species Pseudophialophora oryzae sp. nov based on the molecular phylogeny and morphological characteristics. Fluorescent protein-expressing P. oryzae was used to monitor the fungal colonization pattern. Hyphae invaded the epidermis to the inner cortex but not into the root stele. The inoculation of P. oryzae promoted the rice growth, with the growth parameters of chlorophyll content, shoot height, root length, fresh shoot weight, fresh root weight and dry weight increasing by 24.10, 35.32, 19.35, 90.00, 33.3, and 79.17%, respectively. P. oryzae induced up-regulation of nitrate transporter OsPTR9 and potassium transporter OsHAK16 by 7.28 ± 0.84 and 2.57 ± 0.80 folds, promoting nitrogen and potassium elements absorption. In addition, P. oryzae also conferred a systemic resistance against rice blast, showing a 72.65 and 75.63% control rate in sterile plates and potting conditions. This systemic resistance was mediated by the strongly up-regulated expression of resistance-related genes NAC, OsSAUR2, OsWRKY71, EL5, and PR1α. Since P. oryzae can promote rice growth, biomass and induce systemic disease resistance, it can be further developed as a new biogenic agent for agricultural production, providing a new approach for biocontrol of rice blast.

An efficient genetic manipulation protocol for dark septate endophyte Falciphora oryzae.

OBJECTIVE: To investigate the protoplast preparation and transformation system of endophytic fungus Falciphora oryzae. RESULTS: F. oryzae strain obtained higher protoplast yield and effective transformation when treated with enzyme digestion solution containing 0.9 M KCl solution and 10 mg mL-1 glucanase at 30 °C with shaking at 80 rpm for 2-3 h. When the protoplasts were plated on a regenerations-agar medium containing 1 M sucrose, the re-growth rate of protoplasts was the highest. We successfully acquired green fluorescent protein-expressing transformants by transforming the pKD6-GFP vector into protoplasts. Further, the GFP expression in fungal hyphae possessed good stability and intensity during symbiosis in rice roots. CONCLUSIONS: This study provided a protoplast transformation system of F. oryzae, creating opportunities for future genetic research in other endophytic fungi.

Dark septate endophyte Falciphora oryzae-assisted alleviation of cadmium in rice.

Dark septate endophytes (DSEs) are the typical representatives of root endophytic fungi in heavy metal (HM)-contaminated environments. However, little is known about their roles in the HMs tolerance of hosts and the underlying mechanism. Here, we investigated the biological roles and molecular mechanisms of a DSE strain Falciphora oryzae in alleviating cadmium (Cd) toxicities in rice. It was found that F. oryzae possessed a capacity of accumulating Cd in its vacuoles and chlamydospores. During symbiosis, F. oryzae conferred improved Cd tolerance to rice, decreasing Cd accumulation in roots and translocation to shoots. F. oryzae alleviated Cd toxicity to rice by sequestering Cd in its vacuoles. Further application of F. oryzae as fertilizer in the field could reduce Cd content in rice grains. We identified a SNARE Syntaxin 1 gene through proteomics, which participated in Cd tolerance of F. oryzae by regulating chlamydospore formation and vacuole enlargement. This study provided novel insights into how the DSEs and their host plants combat Cd stress.

Potential role of FoxO3a in the regulation of trophoblast development and pregnancy complications.

The forkhead box O3a protein (FoxO3a) has been reported to regulate tumour invasion and migration, but little is known about the molecular mechanism or its role in trophoblast invasion and migration into the uterus. In this study, we aim to explore its role in trophoblast development and placenta-related pregnancy complications and the potential mechanism. Levels of FoxO3a and its phosphorylated form (p-FoxO3a) in placental tissue from healthy pregnant women and pre-eclampsia patients were first compared. Then, HTR-8/SVneo cells were transfected with lentiviral vectors to deplete and overexpress FoxO3a. Western blot, immunohistochemistry, Cell Counting Kit-8, wound-healing assay, Matrigel invasion assay, cell apoptosis, cell cycle assay, RNA sequencing, qRT-PCR and ChIP-qPCR were performed on the cells to study the potential role of FoxO3a and the underlying mechanism. We found the expression of FoxO3a was decreased, whereas p-FoxO3a was increased in pre-eclampsia placentae. FoxO3a depletion significantly reduced transcription of the promoter region of intercellular cell adhesion molecule-1 (ICAM1) gene in ChIP assays and led to reduced invasion and migration of trophoblast cells, arrested cell cycle in G1 phase and increased apoptosis under oxidative stress. Our results suggested that FoxO3a may play a role in the regulation of trophoblast invasion and migration during placental development, which may be because of its affinity to the ICAM1 promotor.

Iodine-Catalyzed Regioselective Oxidative Cyclization of Aldehyde Hydrazones with Electron-Deficient Olefins for the Synthesis of Mefenpyr-Diethyl.

A regioselective synthesis of polysubstituted dihydropyrazoles and pyrazoles through an iodine-catalyzed oxidative cyclization strategy of aldehyde hydrazones with electron-deficient olefins is described. The protocol adopts very mild reaction conditions and provides desirable yields. The reaction is supposed to proceed via a cascade C-H functionalization, C-N bond formation, and oxidation sequential processes. The overall simplicity and regioselectivity of the catalytic system make this approach a valuable and step-economical tool to construct a C-C bond for the synthesis of Mefenpyr-Diethyl.

Cascade Functionalization of C(sp3)-Br/C(sp2)-H Bonds: Access to Fused Benzo[e]isoindole-1,3,5-trione via Visible-Light-Induced Reductive Radical Relay Strategy.

A reductive radical relay strategy for the construction of fused benzo[e]isoindole-1,3,5-trione through a reaction of α-bromo ketones with maleimides in the presence of Ir(ppy)3 under visible-light irradiation is described. The protocol employs very mild reaction conditions and offers satisfactory yields. Moreover, the reaction proceeds through a cascade C(sp3)-Br/C(sp2)-H functionalization, double C-C bond formation, and oxidative aromatization sequence.

Pyrrolo[3,4- c]pyrazole Synthesis via Copper(Ι) Chloride-Catalyzed Oxidative Coupling of Hydrazones to Maleimides.

A variety of pyrrolo[3,4- c]pyrazole derivatives from readily available aldehyde hydrazones and maleimides via direct oxidative coupling under radical cascade reaction have been reported. This method offers satisfactory chemical yields and good functional group compatibility. Moreover, this practical approach is catalyzed by CuCl utilizing air as the oxidant and some control experiments were performed to elaborate the mechanism.

Copper-Catalyzed Oxidative Cyclization of Maleimides with Amines and Alkyne Esters: Direct Access to Fully Substituted Dihydropyrroles and Pyrrole Derivatives.

An efficient and practical Cu(I)-catalyzed oxidative cyclization cascade reaction of diverse amines, alkyne esters and maleimides has been developed. The reactions can afford 4,6-dioxopyrrolo[3,4-b]pyrrole-2,3-dicarboxylates and related derivatives with satisfactory yields by altering the reaction conditions slightly. The substrate scope highlights the flexibility of the catalyst, and a reaction mechanism is also proposed.