Inhibitory Effect and Mechanism of Carvacrol against Black Mold Disease Agent Alternaria alternata in Goji Berries.

Alternaria alternata, as a main decay fungus of goji berry, can produce mycotoxins such as alternariol (AOH), alternariol monomethyl ether (AME), and tenuazonic acid (TeA). Carvacrol (CVR) has exhibited a broad-spectrum antifungal activity in vitro. We assumed that CVR can also be applied to control Alternaria rot on goji berries and mycotoxins produced by the pathogens. To investigate whether CVR impacts the accumulation of mycotoxins and cell membrane damage of A. alternata, the antifungal activity of CVR on the fungal growth and mycotoxin production was evaluated in this study. The results showed that the minimum inhibitory concentration (MIC) of CVR against A. alternata was 0.12 µL/mL. Meanwhile, the destruction of plasma membrane integrity, cytoplasmic leakage, intracellular oxidative damage, and inhibitory effect in vivo were also observed in A. alternata treated with CVR. Moreover, CVR significantly reduced the accumulation of AOH, AME, and TeA. Transcriptomic profiling was performed by means of comparative RNA-Seq analysis to research the gene expression level of A. alternata, which attested to significant changes in nitrogen metabolism, carbon utilization, fatty acid oxidation, and antioxidant enzymes in CVR-treated A. alternata. This study suggests a new understanding of the molecular mechanism of response to CVR treatment in A. alternata, indicating that CVR is a novel antifungal agent with the potential to be applied to various fungi.

Cell structure damage contributes to antifungal activity of sodium propylparaben against Trichothecium roseum.

Trichothecium roseum is a type of fungus that causes pink rot in muskmelon after the melons are harvested. Pink rot leads to severe decay during storage and causes the production of toxins that can be harmful to human health. Sodium propylparaben (SPP, IUPAC name: sodium; 4-propoxycarbonylphenolate) is an antimicrobial preservative that can be used to treat the inedible parts of fruits in addition to food, medications, and packaging. In this study, the effectiveness of SPP in inhibiting T. roseum was tested, and the inhibition mechanism was investigated. The results show that SPP inhibited the growth and spore germination of T. roseum. The malondialdehyde (MDA) content, propidium iodide staining, alkaline phosphatase (AKP) activity, and calcofluor white (CFW) staining results show that SPP produced a disruption of the cell membrane and cell wall integrity of T. roseum. Scanning and transmission electron microscopy (SEM and TEM, respectively) results also indicate that SPP disrupted the cellular structure of T. roseum. Meanwhile, the large amounts of superoxide anion and hydrogen peroxide in T. roseum accumulated due to the effects of SPP on the activities of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, superoxide dismutase, and decreased catalase. In addition, SPP caused a significant reduction in the incidence rate and disease degree of muskmelon pink rot in vivo. In conclusion, SPP appears to be effective against T. roseum via disruption of the cell membrane and wall. SPP could be used to manage melon pink rot after fruit harvesting because of its disease inhibition effect in vivo.

Inhibitory effect of carvacrol against Alternaria alternata causing goji fruit rot by disrupting the integrity and composition of cell wall.

Goji (Lycium barbarum L.) is a widely planted crop in China that is easily infected by the pathogenic fungus Alternaria alternata, which causes rot after harvest. Previous studies showed that carvacrol (CVR) significantly inhibited the mycelial growth of A. alternata in vitro and reduced Alternaria rot in goji fruits in vivo. The present study aimed to explore the antifungal mechanism of CVR against A. alternata. Optical microscopy and calcofluor white (CFW) fluorescence observations showed that CVR affected the cell wall of A. alternata. CVR treatment affected the integrity of the cell wall and the content of substances in the cell wall as measured by alkaline phosphatase (AKP) activity, Fourier transform-infrared spectroscopy (FT-IR), and X-ray photoelectron spectroscopy (XPS). Chitin and ß-1,3-glucan contents in cells decreased after CVR treatment, and the activities of ß-glucan synthase and chitin synthase decreased. Transcriptome analysis revealed that CVR treatment affected cell wall-related genes in A. alternata, thereby affecting cell wall growth. Cell wall resistance also decreased with CVR treatment. Collectively, these results suggest that CVR may exert antifungal activity by interfering with cell wall construction, leading to impairment of cell wall permeability and integrity.

Metabolic Profiling and Molecular Mechanisms Underlying Melatonin-Induced Secondary Metabolism of Postharvest Goji Berry (Lycium barbarum L.).

Postharvest decay of goji berries, mainly caused by Alternaria alternata, results in significant economic losses. To investigate the effects of melatonin (MLT) on resistance to Alternaria rot in goji berries, the fruits were immersed in the MLT solutions with varying concentrations (0, 25, 50, and 75 µmol L-1) and then inoculated with A. alternata. The results showed that the fruits treated with 50 µmol L-1 MLT exhibited the lowest disease incidence and least lesion diameter. Meanwhile, endogenous MLT in the fruits treated with 50 µmol L-1 MLT showed higher levels than in the control fruits during storage at 4 ± 0.5 °C. Further, the enzymatic activities and expressions of genes encoding peroxidase, phenylalanine ammonia-lyase, cinnamate 4-hydroxylase, 4-coumarate-CoA ligase, chalcone synthase, chalcone isomerase, and cinnamyl alcohol dehydrogenase were induced in the treated fruit during storage. UPLC-ESI-MS/MS revealed that secondary metabolites in the fruits on day 0, in order of highest to lowest levels, were rutin, p-coumaric acid, chlorogenic acid, ferulic acid, caffeic acid, naringenin, quercetin, kaempferol, and protocatechuic acid. MLT-treated fruits exhibited higher levels of secondary metabolites than the control. In conclusion, MLT treatment contributed to controlling the postharvest decay of goji fruit during storage by boosting endogenous MLT levels, thus activating the antioxidant system and secondary metabolism.

Construction of vascularized tissue-engineered bone with polylysine-modified coral hydroxyapatite and a double cell-sheet complex to repair a large radius bone defect in rabbits.

In this study, the potential of vascularized tissue-engineered bone constructed by a double cell-sheet (DCS) complex and polylysine (PLL)-modified coralline hydroxyapatite (CHA) to repair large radius bone defects was investigated in rabbits. Firstly, the DCS complex was obtained after rabbit adipose-derived mesenchymal stem cell (ADSC) culture was induced. Secondly, PLL-CHA composite scaffolds with different concentrations of PLL were prepared by the soaking and vacuum freeze-drying methods, and then the scaffolds were characterized by X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, compression performance testing and cytocompatibility evaluation. Thirdly, DCS-PLL-CHA vascularized tissue-engineered bone was constructed in vitro and transplanted into a large radius bone defect model in rabbits. Finally, the potential of the DCS-PLL-CHA vascularized tissue-engineered bone to repair the large bone defect was evaluated through general observations, laser speckle imaging, scanning electron microscopy (SEM), histological staining, radiography observations and RT-PCR. The in vitro experimental results showed that the DCS complex provided a very large cell reserve, which carried a large number of osteoblasts and vascular endothelial cells that were induced in vitro. When the DCS complex was combined with the PLL-CHA scaffold in vitro, the effects of PLL on cell adhesion, proliferation and differentiation led to a situation similar to the chemotaxis of the body, making the combined complex more conducive to graft cellularization than the DCS complex alone. The in vivo experiments showed blood supply on the surface of the callus in each group, and the amount of blood perfusion on the surface of the defect area was almost equal among the groups. At 12 weeks, the surface of the DCS-PLL-CHA group was completely wrapped by bone tissue and osteoids, the cortical bone image was basically continuous, and the medullary cavity was mainly perforated. A large amount of well-arranged lamellar bone was formed, a small amount of undegraded CHA exhibited a linear pattern, and a large amount of bone filling could be seen in the pores. At 12 weeks, the expression levels of BGLAP, SPP1 and VEGF were similar in each group, but PECAM1 expression was higher in the DCS-PLL-CHA group than in the autogenous bone group and CHA group. The results showed that PLL could effectively promote the adhesion, proliferation and differentiation of ADSCs and that DCS-PLL-CHA vascularized tissue-engineered bone has potential for bone regeneration and bone reconstruction and can be used to repair large bone defects. STATEMENT OF SIGNIFICANCE: 1. PLL-CHA composite scaffolds with different concentrations of PLL were prepared by the soaking and vacuum freeze-drying methods. 2. The vascularized tissue-engineered bone was constructed by the double cell sheet (DCS) complex combined with PLL-CHA scaffolds. 3. The DCS-PLL-CHA vascularized tissue-engineered bone has potential for bone regeneration and bone reconstruction and can be used to repair large bone defects.

Construction of vascularized tissue-engineered bone with a double-cell sheet complex.

A double-cell sheet (DCS) complex composed of an osteogenic cell sheet and a vascular endothelial cell sheet with osteogenesis and blood vessel formation potential was developed in this study. The osteogenic and vascular endothelial cell sheets were obtained after induced culture of rabbit adipose-derived mesenchymal stem cells. The osteogenic cell sheet showed positive alizarin red, von Kossa, and alkaline phosphatase (ALP) staining. The vascular endothelial cell sheet exhibited visible W-P bodies in the cells, the expression of CD31 was positive, and a vascular mesh structure was spontaneously formed in a Matrigel matrix. The subcutaneous transplantation results for four groups of DCS and DCS-coral hydroxyapatite (CHA) complexes, and the CHA scaffold group in nude mice revealed mineralization of collagen fibers and vascularization in each group at 12 weeks, but the degrees of mineralization and vascularization showed differences among groups. The pattern involving endothelial cell sheets covered with osteogenic cell sheets, group B, exhibited the best results. In addition, the degree of mineralization of the DCS-CHA complexes was more mature than those of the same group of DCS complexes and the CHA scaffold, and the capillary number was greater than those of the same group of DCS complexes and the CHA scaffold. Therefore, the CHA scaffold strengthened the osteogenesis and blood vessel formation potential of the DCS complexes. Meanwhile, the DCS complexes also promoted the osteogenesis and blood vessel formation potential of the CHA scaffold. This study will provide a basis for building vascularized tissue-engineered bone for bone defect therapy. STATEMENT OF SIGNIFICANCE: This study developed a double-cell sheet (DCS) complex composed of an osteogenic cell sheet and a vascular endothelial cell sheet with osteogenesis and blood vessel formation potential. Osteogenic and vascular endothelial cell sheets were obtained after induced culture of rabbit adipose-derived mesenchymal stem cells. The DCS complex and DCS-CHA complex exhibited osteogenic and blood vessel formation potential in vivo. CHA enhanced the osteogenesis and blood vessel formation abilities of the DCS complexes in vivo. Meanwhile, the DCS complexes also promoted the osteogenesis and blood vessel formation potential of the CHA scaffold. Group B of the DCS complexes and DCS-CHA complexes exhibited the best osteogenesis and blood vessel formation abilities.

Construction and characterization of osteogenic and vascular endothelial cell sheets from rat adipose-derived mesenchymal stem cells.

In this study, adipose-derived mesenchymal stem cells (ADSCs) were isolated from adipose tissues of rats. Flow cytometry identification showed that ADSCs of passage 3 highly expressed CD29 and CD44, but hardly expressed CD31 and CD45. Adipogenic, osteogenic, and chondrogenic differentiation were confirmed by the results of oil red O staining, alkaline phosphatase (ALP), and alcian blue staining, respectively. ADSCs at a density of 1×10(6)/cm(2) were cultured in the osteogenic medium and the osteogenic cell sheets could be obtained after 14 d. The cell sheets were positive with von kossa staining. The transmission electron microscopy (TEM) result showed that needle-like calcium salt crystals were deposited on the ECM. These results suggested that the osteogenic cell sheets may have potential osteogenesis ability. ADSCs at a density of 1×10(6)/cm(2) were cultured in the endothelial cell growth medium-2 and the endothelial cell sheets can be formed after 16 d of culture. The TEM image confirmed that the Weibel-Palade corpuscle was seen in the cells. The expression of CD31 was positive, suggesting that the endothelial cell sheets may have a strong ability to form blood vessels. In this study, two types of cell sheets with the potential abilities of osteogenesis and blood vessels formation were obtained by induced culture of ADSCs in vitro, which lays a foundation to build vascularized tissue engineered bone for the therapy of bone defects.

Bilayered PLGA/Wool Keratin Composite Membranes Support Periodontal Regeneration in Beagle Dogs.

Regenerative procedures using guided tissue regeneration (GTR) membrane are presently well-established in periodontal therapy. In this study, bilayered poly(lactic-co-glycolic acid) (PLGA)/wool keratin (WK) composite membranes were prepared for use as GTR membrane by the solvent casting and electrospinning methods. Then, the composite membranes were used to evaluate the effects in guided tissue regeneration in beagle dogs. The results showed that the bottom layer (casting film) of the bilayered PLGA/wool keratin composite membranes was a compact PLGA/wool keratin membrane, and the upper layer (electrospun film) was a loose, porous, three-dimensional, and fibrous PLGA/wool keratin membrane (The wool keratin was at five different levels: 0, 0.5, 1, 2, or 4 wt %). The mechanical properties of the composites were significantly enhanced by the addition of wool keratin. The bilayered PLGA/1.0% wool keratin composite membranes presented the best values of ultimate strength and Young's modulus. All of the bilayered PLGA/wool keratin composite membranes showed high thermal and thermooxidative stabilities. The GTR results showed that the PLGA/1.0% wool keratin composite membranes could effectively promote the periodontal tissue regeneration after 12 weeks, and have a similar effect to the collagen membrane on the regeneration of the periodontal tissue. Thus, the bilayered PLGA/wool keratin composite membranes show great potential to meet the demand for GTR membrane. The study will serve as a foundation for further study of the PLGA/wool keratin membranes for GTR application and the therapy of periodontal disease.