The essential oil extracted from the seeds of dill (Anethum graveolens L.) was demonstrated in this study as a potential source of an eco-friendly antifungal agent. To elucidate the mechanism of the antifungal action further, the effect of the essential oil on the plasma membrane and mitochondria of Aspergillus flavus was investigated. The lesion in the plasma membrane was detected through flow cytometry and further verified through the inhibition of ergosterol synthesis. The essential oil caused morphological changes in the cells of A. flavus and a reduction in the ergosterol quantity. Moreover, mitochondrial membrane potential (MMP), acidification of external medium, and mitochondrial ATPase and dehydrogenase activities were detected. The reactive oxygen species (ROS) accumulation was also examined through fluorometric assay. Exposure to dill oil resulted in an elevation of MMP, and in the suppression of the glucose-induced decrease in external pH at 4 µl/ml. Decreased ATPase and dehydrogenase activities in A. flavus cells were also observed in a dose-dependent manner. The above dysfunctions of the mitochondria caused ROS accumulation in A. flavus. A reduction in cell viability was prevented through the addition of L-cysteine, which indicates that ROS is an important mediator of the antifungal action of dill oil. In summary, the antifungal activity of dill oil results from its ability to disrupt the permeability barrier of the plasma membrane and from the mitochondrial dysfunction-induced ROS accumulation in A. flavus.

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We investigated the potential of human hair follicle cells for multilineage differentiation and as a source of functional smooth muscle cells (SMCs). We report that human hair follicle stem cells (HFCs) isolated from individual follicles expressed surface markers that are characteristic of mesenchymal stem cells such as CD44, CD49b, CD73, CD90, and CD105 but lacked hematopoietic markers CD45 and CD34. In addition, HFCs differentiated toward adipocytes, chondrocytes, osteoblasts, or SMCs in the appropriate induction medium. Treatment with basic fibroblast growth factor increased proliferation and prevented myogenic differentiation, suggesting that basic fibroblast growth factor can be used to expand the population of undifferentiated HFCs to the large numbers needed for therapeutic applications. SMCs were isolated from HFCs using tissue-specific promoters and flow cytometry sorting. Cylindrical vascular constructs engineered with HF-SMCs showed remarkable contractility in response to receptor and nonreceptor agonists such KCl, endothelin-1, and the thromboxane mimetic, U46619, as well as superior mechanical properties compared to their counterparts with human vascular SMCs. Our results suggest that HF is a rich source of mesenchymal stem cells with great potential for myogenic differentiation providing functional SMCs for tissue regeneration and cell therapies.

In the present study, we provide detailed insights into perillaldehyde (PAE)'s mechanisms of action on Aspergillus flavus and offer evidence in favor of the induction of an apoptosis-like phenotype. Specifically, PAE's antifungal mode of action was investigated through the detection of mitochondrial membrane potential (MtΔψ) and phosphatidylserine (PS) exposure, as well as intracellular Ca level, reactive oxygen species accumulation, and metacaspase activation. This was done by way of fluorometry, measuring DNA fragmentation, and condensation by fluorescent microscopy. Furthermore, we searched for phenotypic changes characteristic of apoptosis by transmission electron microscopy and flow cytometry, determining the amount of cytochrome c released using Western blotting. Results indicated that cultivation of A. flavus in the presence of PAE caused depolarization of MtΔψ, rapid DNA condensation, large-scale DNA fragmentation, and an elevation of intracellular Ca level. The percentage of early apoptotic cells with exposure of PS were 27.4% and 48.7%, respectively, after 9 h incubations with 0.25 and 0.5 μL/mL of PAE. The percentage of stained cells with activated intracellular metacaspases exposed to PAE at concentrations of 0.25 and 0.5 μL/mL compared with control subjects were increased by 28.4 ± 3.25% and 37.9 ± 4.24%, respectively. The above results has revealed that PAE induces fungal apoptosis through a caspase-dependent mitochondrial pathway. In all, our findings provide a novel mechanism for exploring a possible antifungal agent used in food preservation.

Expression of multiple genes from the same target cell is required in several technological and therapeutic applications such as quantitative measurements of promoter activity or in vivo tracking of stem cells. In spite of such need, reaching independent and high level dual-gene expression cannot be reliably accomplished by current gene transfer vehicles. To address this issue, we designed a lentiviral vector carrying two transcriptional units separated by polyadenylation, terminator and insulator sequences. With this design, the expression level of both genes was as high as that yielded from lentiviral vectors containing only a single transcriptional unit. Similar results were observed with several promoters and cell types including epidermal keratinocytes, bone marrow mesenchymal stem cells and hair follicle stem cells. Notably, we demonstrated quantitative dynamic monitoring of gene expression in primary cells with no need for selection protocols suggesting that this optimized lentivirus may be useful in high-throughput gene expression profiling studies.