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Information on the reproductive anatomy in genera of the tribe Naucleeae, particularly Cephalanthus, is scarce and fragmented. Of the six species in the genus, only the mature megagamethophyte of Cephalanthus occidentalis has been described. This study aims to provide information on embryological aspects in flowers of C. glabratus and to analyze the morphology and anatomy of the flowers, fruit, and seed in the six species of the genus. Cephalanthus glabratus have imperfect flowers pistillate (PF) and staminate (SF). In the PF, the ovules are functional, while in the SF, they atrophy during the formation of the embryo sac. The mature ovule has a single integument, corresponds to the Phyllis type and the embryo sac is a Polygonum type, forming only in the PF. The presence of pollenkitt and secondary presentation of pollen were observed in the SF, as well as in the pollen formation previously described, whereas in the PF, they are absent, due to the collapse of the pollen grains inside the indehiscent anthers. The analysis of the ontogeny of the ovular excrescence in C. glabratus determined its funicular origin, calling it an aril. Its development is a pre-anthesis event, initiated during megasporogenesis. In seeds, the aril is a fleshy, white appendage which almost completely envelops the seeds of Cephalanthus, except for Cephalanthus natalensis where it is noticeably more reduced. Studies of the fruit in Cephalanthus species indicate that the infructescence is a dry schizocarp which separates into uni-seminated mericarps, except in C. natalensis that has fleshy indehiscent fruit.Gamma-aminobutyric acid (GABA), an important bioactive compound, is synthesized through the decarboxylation of L-glutamate (L-Glu) by glutamate decarboxylase (GAD). The use of lactic acid bacteria (LAB) as catalysts opens interesting avenues for the biosynthesis of food-grade GABA. However, a key obstacle involved in the improvement of GABA production is how to resolve the discrepancy of optimal pH between the intracellular GAD activity and cell growth. In this work, a potential GAD candidate (LpGadB) from Lactobacillus plantarum was heterologously expressed in Escherichia coli. Recombinant LpGadB existed as a homodimer under the native conditions with a molecular mass of 109.6 kDa and exhibited maximal activity at 40°C and pH 5.0. selleck The Km value and catalytic efficiency (kcat/Km) of LpGadB for L-Glu was 21.33 mM and 1.19 mM-1s-1, respectively, with the specific activity of 26.67 μM/min/mg protein. Subsequently, four C-terminally truncated LpGadB mutants (GadBΔC10, GadBΔC11, GadBΔC12, GadBΔC13) were constructed based on homology modeling. Among them, the mutant GadBΔC11 with highest catalytic activity at near-neutral pH values was selected. In further, the GadBΔC11 and Glu/GABA antiporter (GadC) of Lactococcus lactis were co-overexpressed in the host L. lactis NZ3900. Finally, after 48 h of batch fermentation, the engineered strain L. lactis NZ3900/pNZ8149-gadBΔC11C yielded GABA concentration up to 33.52 g/L by applying a two-stage pH control strategy. Remarkably, this is the highest yield obtained to date for GABA from fermentation with L. lactis as a microbial cell factory.Key points• The GadB from L. plantarum was heterologously expressed in E. coli and biochemically characterized.• Deletion of the C-plug in GadB shifted its pH-dependent activity toward a higher pH.• Reconstructing the GAD system of L. lactis is an effective approach for improving its GABA production.Natural killer-92 cells (NK-92 cells) need to be efficiently expanded by serum-free culture in vitro to meet clinical requirements. Fatty acids mainly provide substrates for energy production, which is of crucial importance to meet the energy demands of highly proliferating cells. This study optimized the medium (EM) for NK-92 cells by designing an experiment to expand cells efficiently. EM, an in-house designed chemically defined serum-free medium, was used as the basal medium. Fatty acids as additive ingredients were screened and optimized by the experimental design method. Three additives, arachidonic acid, myristic acid and palmitoleic acid, were screened; therefore, the optimized medium was named EM-FA. The total cell expansion of NK-92 cells in EM-FA was 72.61±11.95-fold on day 8, which was significantly higher than the 28.55±8.67-fold expansion in EM. To explore the mechanism by which fatty acids promote NK-92 cell expansion, the cell growth kinetics and metabolic characteristics in EM-FA were analyzed. The results showed that NK-92 cells in EM-FA were rapidly expanded while maintaining their cell phenotype and cytotoxicity and enhancing the oxygen consumption rate and mitochondrial function. Fatty acids promoted ATP production to elevate the energy flux for better cell expansion. This study developed an expansion strategy of NK-92 cells in vitro to facilitate their clinical application. KEY POINTS • Arachidonic acid, myristic acid and palmitoleic acid in serum-free medium were optimized by experimental design to enable the rapid expansion of NK-92 cells in vitro. • Fatty acids upregulated oxidative phosphorylation levels and improved the energy metabolism of NK-92 cells.Enterococcus faecium WEFA23 was previously found effectively against adherence and colonization of Listeria monocytogenes CMCC54007, which might be closely related to its surface layer protein (SLP). In this study, the protective of SLP of E. faecium WEFA23 against infection of L. monocytogenes CMCC54007 was systemically investigated. In vitro assay showed that SLP actively inhibited L. monocytogenes internalization into Caco-2 cell line, with decreasing mRNA level of pro-inflammation cytokines and virulence factors and restoring destroyed intestinal barrier. In vivo assay through excluding SLP of E. faecium WEFA23 by 5 M LiCl represented that SLP increased body weight, reduced mortality and cell counts of L. monocytogenes CMCC54007 in tissues of mice. Further researches showed that SLP protected against L. monocytogenes CMCC54007 infection by modulation of intestinal permeability and immunity, namely, it decreased fluorescein isothiocyanate (FITC)-Dextran in serum, ameliorated destroyed colon structure, and increased number of goblet cells and protein level of TJ protein (Claudin-1, Occludin, and ZO-1) in colon.
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