Fitc-FA-FMK

Propionic acid (PPA) is a weak acid that has been used in food products as a preservative because of its inhibitory effect on microorganisms. In the present study, we investigated the PPA fungal killing mechanism, which showed apoptotic features. First, reactive oxygen species (ROS) accumulation and metacaspase activation were detected by 2',7'-dichlorodihydrofluorescein diacetate and CaspACE FITC-VAD-FMK staining, respectively. Increased fluorescence intensities were observed following exposure to PPA, indicating that PPA produced an oxidative environment through the generation of ROS and activation of metacaspase, which can promote apoptosis signaling. We also examined phosphatidylserine externalization (an early apoptosis marker) and DNA and nuclear fragmentation (late apoptosis markers) after exposure to PPA. Based on the results, we determined that PPA exerts its antifungal effect by inducing apoptotic cell death. Moreover, three additional mitochondrial experiments showed mitochondrial membrane depolarization, calcium accumulation and cytochrome c release after cells were exposed to PPA, indicating that the PPA-induced apoptosis pathway is mediated by mitochondria. In conclusion, PPA induces fungal cell death through mitochondria-mediated apoptosis. Results of this study contribute to a deeper understanding of the preservative effects of PPA.

Oocyte and embryo cryopreservation has been applied successfully in many mammalian species. Nevertheless, pig oocytes, because of their greater susceptibility to cryoinjuries, have shown a reduced ability to be fertilized in vitro and a lower developmental competence. The aim of this study was to evaluate the apoptotic status of porcine oocytes vitrified by Cryotop method. We assessed three parameters linked to the activation of the apoptotic cascade: the exteriorization of phosphatidylserine using Annexin V, the caspase activation using FITC-VAD-FMK and the alteration of plasma membrane permeability employing YO-PRO-1. These assays were performed on control oocytes, oocytes exposed to vitrification solutions (toxicity control) and vitrified oocytes either immediately after warming or after incubation for 2h into maturation medium. The exposition to vitrification solutions triggered an increase of the percentage of oocytes both faintly (VAD+ PI-) and strongly (VAD++ PI-) labeled by FITC-VAD-FMK but not a significant modification of the number of oocytes Annexin V (A+ PI-, early apoptotic) and YO-PRO-1(YP+ PI-, apoptotic) positive in comparison with control oocytes. Oocytes submitted to the whole vitrification procedure showed a rise of the percentage of early apoptotic oocytes (A+ PI-) and FITC-VAD-FMK positive oocytes (VAD+/VAD++ PI-) and a contemporaneous increase of the number of dead oocytes (PI+). On the contrary, vitrified oocytes analyzed immediately after warming did not show a significant increase in the percentage of apoptotic oocytes (YO-PRO-1+/PI-) as compared with the control. Post warming incubation for 2h into maturation medium, in comparison with oocytes analyzed immediately after warming, did not induce any increase in the percentage of early apoptotic (A+ P-) oocytes while a decrease of the percentage of VAD+/PI- oocytes and a contemporaneous increase of VAD-/PI- oocytes were observed. Moreover, the post-warming incubation induced a rise of the percentage of apoptotic oocytes (YO-PRO-1+/PI-). All these data confirm the involvement of apoptotic mechanisms on the injuries induced by vitrification procedure in pig oocytes; explanation of this phenomenon could be useful to improve oocytes' cryopreservation protocols.

Objective:To explore the effect and mechanism of action of bufalin in triple-negative breast cancer (TNBC) drug-resistant cell lines.Methods:The normal human mammary epithelial cell line, TNBC cell line, TNBC adriamycin-resistant cell line, and TNBC docetaxel-resistant cell line were treated with different doses of bufalin (0-1,000 nmol/L) at different time points (0-72 h). Propidium iodide staining, AV-FITC/PI double staining, Hoechst 33342/PI double staining and transmission electron microscopy (TEM) were used to evaluate the death patterns of the cell lines.Results:Bufalin killed the TNBC cell line and its drug-resistant cell lines in a dose/time-dependent manner (all P<0.01). After treatment with bufalin for 24 h, the adriamycin-resistant cell line showed a co-existing pattern of necroptosis and apoptosis. However, at 48 h, necroptosis was the main manifestation. After treatment with bufalin, the expressions of tumor necrosis factor α, phospho-tumor necrosis factor receptor 1, phospho-receptor interacting protein 1 and c-caspase 3 increased (all P<0.01), the killing effect of bufalin could be mostly inhibited by NEC-1, and by z-VAD-fmk (both P<0.01). Besides, the intracellular reactive oxygen species (ROS) levels increased considerably (P<0.01), the antioxidant N-acetyl cysteine or Nec-1 could inhibit the increase of ROS level and the killing effect of bufalin (all P<0.01). The adriamycin-resistant cell line exhibited necroptosis characteristic after 48 h of bufalin treatment under TEM.Conclusions:Bufalin could induce necroptosis through RIP1/ROS-mediated pathway to kill the drug-resistant TNBC cell lines. This finding provides critical experimental data and theoretical basis for the clinical application of bufalin to overcome the difficulties in the treatment of TNBC.

Aims:Indole-3-carbinol (I3C) is a natural compound derived from brassica vegetables, displaying antibacterial activity. The study aims to elucidate the antibacterial mode of action(s) induced by indole-3-carbionol in Escherichia coli and enhance the understandings on the respective contribution of each reactive oxygen species (ROS), superoxide anion (O2-), hydrogen peroxide (H2O2), hydroxyl radical (OH-) during the process.Main methods:The antibacterial activity of I3C was assessed through kinetic assay. The generation of ROS was measured by flow cytometer using H2DCFDA dye, while further analysis of respective contribution was done through application of each scavenger: tiron, thiourea and sodium pyruvate. DNA fragmentation and chromatin condensation were observed by TUNEL and DAPI staining agent. Finally, Annexin V/PI, FITC-VAD-FMK and DiBAC4(3) was applied for detection of apoptosis-like death.Key findings:I3C exhibited antibacterial activity in E. coli through accumulation of ROS and DNA damage, eventually leading to apoptosis-like death. Contribution of each ROS displayed respective manner, OH-exerting the most potent influence whereas O2-showed least impact.Significance:Our study is the first to link I3C to the bacterial apoptosis-like death and displays the potential of this agent as a candidate for potential drugs that could help regulating the E. coli, an opportunistic human pathogen. Moreover, the study focused on investigating the individual contribution of each ROS during the process, trying to enhance the understanding regarding ROS and cellular processes followed by oxidative stress in bacteria.