Synergistic antifungal mechanism of cinnamaldehyde and nonanal against Aspergillus flavus and its application in food preservation.

Aspergillus flavus colonization on agricultural products during preharvest and postharvest results in tremendous economic losses. Inspired by the synergistic antifungal effects of essential oils, the aims of this study were to explore the mechanism of combined cinnamaldehyde and nonanal (SCAN) against A. flavus and to evaluate the antifungal activity of SCAN loading into diatomite (DM). Shriveled mycelia were observed by scanning electron microscopy, especially in the SCAN treatment group. Calcofluor white staining, transmission electron microscopy, dichloro-dihydro-fluorescein diacetate staining and the inhibition of key enzymes in tricarboxylic acid cycle indicated that the antifungal mechanism of SCAN against A. flavus was related to the cell wall damage, reactive oxygen species accumulation and energy metabolism interruption. RNA sequencing revealed that some genes involved in antioxidation were upregulated, whereas genes responsible for cell wall biosynthesis, oxidative stress, cell cycle and spore development were significantly downregulated, supporting the occurrence of cellular apoptosis. In addition, compared with the control group, conidia production in 1.5 mg/mL DM/cinnamaldehyde, DM/nonanal and DM/SCAN groups were decreased by 27.16%, 48.22% and 76.66%, respectively, and the aflatoxin B1 (AFB1) contents decreased by 2.00%, 73.02% and 84.15%, respectively. These finding suggest that DM/SCAN complex has potential uses in food preservation.

Sub3 inhibits Aspergillus flavus growth by disrupting mitochondrial energy metabolism, and has potential biocontrol during peanut storage.

BACKGROUND: Aspergillus flavus, a saprophytic fungus, is regularly detected in oil-enriched seeds. During colonization, this organism releases aflatoxins that pose a serious risk to food safety and human health. Therefore, an eco-friendly biological approach to inhibit the pathogen is desirable. RESULTS: Experimental results indicated that A. flavus spores could not germinate in potato dextrose broth culture medium, when the concentration of Sub3 exceeded 0.15 g L-1 . Morphological evaluation performed by flow cytometry and scanning electron microscopy indicated that spores were shrunken and pitted following Sub3 exposure. Physiological assessment using propidium iodide, 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide, 2,7-dichlorodihydrofluorescein diacetate and 4',6-diamidino-2-phenylindole staining revealed damaged cell membranes, decreased mitochondrial membrane potential, increased intracellular reactive oxygen species levels, and elevated large nuclear condensation and DNA fragmentation. Moreover, mitochondrial dehydrogenase activity was reduced by 29.42% and 45.48% after treatment with 0.1 and 0.15 g L-1 Sub3, respectively. Additionally, colonization capacity in peanut was significantly decreased, and the number of spores on seeds treated with Sub3 was decreased by 26.86% (0.1 g L-1 ) and 77.74% (0.15 g L-1 ) compared with the control group. CONCLUSION: Sub3 likely inhibits A. flavus by crossing the cell wall and targeting the cell membrane, disrupting mitochondrial energy metabolism, and inducing DNA damage, leading to spore death. Thus, Sub3 may provide a useful biocontrol strategy to control A. flavus growth in peanuts. © 2020 Society of Chemical Industry.

Expression and purification of recombinant puroindoline A protein in Escherichia coli and its antifungal effect against Aspergillus flavus.

Aspergillus flavus is the main cause of postharvest agricultural commodity loss. In this study, puroindoline A (PINA) protein was expressed in Escherichia coli, purified, and its antifungal properties against A. flavus were characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that the molecular weight of the recombinant PINA protein was approximately 44 kDa. PINA exerted a powerful antifungal effect against A. flavus at 42.42 µg/mL on potato dextrose agar culture medium. Flow cytometry and scanning electron microscopy revealed that the spore morphology was damaged by PINA exposure; spores were depressed and broken, suggesting that the cell wall was impaired. Transmission electron microscopy and propidium iodide staining illustrated significant changes in intracellular spore structure, indicating cell membrane damage. 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide staining indicated decreased mitochondrial membrane potential. Large nuclear condensation and DNA fragmentation were detected by 4',6-diamidino-2-phenylindole staining. The expression of genes related to the cell wall, cell membrane, and spore germination significantly changed in PINA-treated cells; this illustrated the probable mode of PINA action on A. flavus through cell wall destruction and triggered cell membrane, mitochondrial, and DNA damage leading to cell death. The antifungal mechanism of wheat PINA protein on A. flavus has been demonstrated in this study, and has potential application in preventing postharvest loss in the agricultural industry.

Twist contributes to proliferation and epithelial-to-mesenchymal transition-induced fibrosis by regulating YB-1 in human peritoneal mesothelial cells.

Twist is overexpressed in high glucose (HG) damage of human peritoneal mesothelial cells (HPMCs) in vitro. Herein, we further identified its precise function related to fibrosis of peritoneal membranes (PMs). The overexpression and activation of Twist and YB-1 (official name, YBX1) and a transformed fibroblastic phenotype of HPMCs were found to be positively related to epithelial-mesenchymal transition progress and PM fibrosis ex vivo in 93 patients who underwent continuous ambulatory peritoneal dialysis (PD), and also in HG-induced immortal HPMCs and an animal model of PD. Evidence from chromatin immunoprecipitation and luciferase reporter assays supported that YBX1 is transcriptionally regulated by the direct binding of Twist to E-box. Overexpression of Twist and YB-1 led to an increase in epithelial-mesenchymal transition, proliferation, and cell cycle progress of HPMCs, which might contribute to PM fibrosis. In contrast, the silencing of Twist or YB-1 inhibited HG-induced growth and cell cycle progression of HPMCs; this led to a down-regulation in the expression of cyclin Ds and cyclin-dependent kinases, finally inhibiting PM fibrosis. Twist contributes to PM fibrosis during PD treatment, mainly through regulation of YB-1.

Innovative fluorescent magnetic albumin microbead-assisted cell labeling and intracellular imaging of glioblastoma cells.

Superparamagnetic nanoparticle-based polymer microbeads utilized as carriers are attractive materials widely applied in the biomedical field. However, the deficiency of toxicity, biocompatibility, and biodegradability for polymer materials often limits the application of these microbeads. In the present study, magnetic albumin microbeads (MAMbs), i.e., human serum albumin-coated γ-Fe2O3 nanoparticles, are synthesized to label human U251 glioblastoma multiforme cells. The effects of MAMbs on the biological behavior of U251 glioblastoma cells, including their proliferation, cell viability, cytoskeletal structure, cell cycle, and apoptosis rate, are investigated. Moreover, fluorescein isothiocyanate (FITC)-MAMbs are fabricated by reaction with fluorescent dye FITC used for intracellular imaging of U251 glioblastoma cells. MAMbs possess undetectable cytotoxicity and excellent biocompatibility with U251 glioblastoma cells, as demonstrated by the biological behavior and morphology of U251 cells exposed to MAMbs. Furthermore, the constructed fluorescent MAMbs allow effective intracellular imaging, as illustrated by fluorescence microscopic analysis. The fabricated fluorescent MAMbs have promising perspectives in biomedical research, especially in cell-targeted labeling and intracellular fluorescence magnetic dual-mode imaging in cancer-targeted diagnosis and therapy.

Twist overexpression promoted epithelial-to-mesenchymal transition of human peritoneal mesothelial cells under high glucose.

BACKGROUND: Long-term peritoneal dialysis (PD) results in functional and structural alterations of the peritoneal membrane. Previous studies have suggested that high glucose (HG) could induce transdifferentiation of peritoneal mesothelial cells into myofibroblasts, but the molecular mechanisms of HG-induced epithelial-to-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs) are unclear. This study was undertaken to elucidate the effects and mechanisms of Twist on HG-induced EMT of HPMCs. METHODS: HPMCs were exposed to 5.6 mM glucose [normal glucose (NG)], 50 mM glucose (HG) or 50 mM glucose with Si-Twist or pcDNA3.1-Twist. Western blot and immuocytochemistry were performed to determine Twist, E-cadherin and α-smooth muscle actin (α-SMA) protein expression. MMP2 and MMP9 were detected by zymography. Rats were daily instilled with PD fluid and lipopolysaccharide (LPS) or sodium chloride during 6 weeks. Histological analyses were carried out in parietal peritoneum. Twist was detected by western blotting. RESULTS: Twist and α-SMA protein and immuocytochemistry were significantly increased in HG-conditioned media compared to NG media. E-cadherin protein was lower in pcDNA3.1-Twist-transfected HPMCs compared to pcDNA3.1 cells. Twist protein was upregulated 12 h after HG stimulation. MMP9 was increased in pcDNA3.1-Twist-transfected HPMCs compared to pcDNA3.1 cells. Exposure of rat peritoneum to PD fluid and LPS resulted in an increase of extracellular matrix deposition. Twist and α-SMA were stained in the PD fluid group and compared to the control group. Twist protein was significantly increased in the PD group. CONCLUSIONS: In conclusion, HG-induced Twist expression might contribute to EMT of HPMCs. Twist may control EMT of HPMCs by regulating MMP9.

[Improved culturability of soil bacteria using proper combination with various culturing medium].

To isolate more unique and previously unrecognized bacteria in soil samples, the culture difference under three incubation modes was investigated by using trophic, low-nutrient broth and soil extract as growth medium. Plate count proved that the oligotrophic medium resulted in a slow growth and consecutive colony formation over the course of incubation. On the 5th day, the most number of colony-forming unit was found on trophic LB and low-nutrient R2A, which was approximate 5 times as many as that isolated on 0.1 x LB. Of the 7 media, LB broth harvested the maximum bacterial communities, and novel species could be isolated as the nutrient was diluted to appropriate extent. The DGGE patterns of oligotrophic and rich nutrient culture collection displayed low similarity, however, the bands at various lanes exhibited complementary effect. When cultivated with static flask, LB and R2A media obtained more bacterial species, which concluded most species isolated by the other five media. Under the test tube incubation mode, the most species was also found in LB medium except some appeared only on R2A and TSB. Apparent bacterial communities difference could be detected between R2A, LB and TSB media. The experiment data may contribute much to the special medium design as well as improvement of bacterial culturability by using proper medium.