Atmospheric-Pressure Plasma Jet Induces Apoptosis Involving Mitochondria via Generation of Free Radicals

The plasma jet has been proposed as a novel therapeutic method for anticancer treatment. However, its biological effects and mechanism of action remain elusive. Here, we investigated its cell death effects and underlying molecular mechanisms, using air and N2 plasma jets from a micro nozzle array. Treatment with air or N2 plasma jets caused apoptotic death in human cervical cancer HeLa cells, simultaneously with depolarization of mitochondrial membrane potential. In addition, the plasma jets were able to generate reactive oxygen species (ROS), which function as surrogate apoptotic signals by targeting the mitochondrial membrane potential. Antioxidants or caspase inhibitors ameliorated the apoptotic cell death induced by the air and N2 plasma jets, suggesting that the plasma jet may generate ROS as a proapoptotic cue, thus initiating mitochondria-mediated apoptosis. Taken together, our data suggest the potential employment of plasma jets as a novel therapy for cancer

Deregulation of CREB Signaling Pathway Induced by Chronic Hyperglycemia Downregulates NeuroD Transcription

CREB mediates the transcriptional effects of glucose and incretin hormones in insulin-target cells and insulin-producing β-cells. Although the inhibition of CREB activity is known to decrease the β-cell mass, it is still unknown what factors inversely alter the CREB signaling pathway in β-cells. Here, we show that β-cell dysfunctions occurring in chronic hyperglycemia are not caused by simple inhibition of CREB activity but rather by the persistent activation of CREB due to decreases in protein phophatase PP2A. When freshly isolated rat pancreatic islets were chronically exposed to 25 mM (high) glucose, the PP2A activity was reduced with a concomitant increase in active pCREB. Brief challenges with 15 mM glucose or 30 µM forskolin after 2 hour fasting further increased the level of pCREB and consequently induced the persistent expression of ICER. The excessively produced ICER was sufficient to repress the transcription of NeuroD, insulin, and SUR1 genes. In contrast, when islets were grown in 5 mM (low) glucose, CREB was transiently activated in response to glucose or forskolin stimuli. Thus, ICER expression was transient and insufficient to repress those target genes. Importantly, overexpression of PP2A reversed the adverse effects of chronic hyperglycemia and successfully restored the transient activation of CREB and ICER. Conversely, depletion of PP2A with siRNA was sufficient to disrupt the negative feedback regulation of CREB and induce hyperglycemic phenotypes even under low glucose conditions. Our findings suggest that the failure of the negative feedback regulation of CREB is the primary cause for β-cell dysfunctions under conditions of pathogenic hyperglycemia, and PP2A can be a novel target for future therapies aiming to protect β-cells mass in the late transitional phase of non-insulin dependent type 2 diabetes (NIDDM)

Antimicrobial Effects of a Hexapetide KCM21 against Pseudomonas syringae pv. tomato DC3000 and Clavibacter michiganensis subsp. michiganensis

Antimicrobial peptides (AMPs) are small but effective cationic peptides with variable length. In previous study, four hexapeptides were identified that showed antimicrobial activities against various phytopathogenic bacteria. KCM21, the most effective antimicrobial peptide, was selected for further analysis to understand its modes of action by monitoring inhibitory effects of various cations, time-dependent antimicrobial kinetics, and observing cell disruption by electron microscopy. The effects of KCM21 on Gram-negative strain, Pseudomonas syringae pv. tomato DC3000 and Gram-positive strain, Clavibacter michiganensis subsp. michiganensis were compared. Treatment with divalent cations such as Ca²⁺ and Mg²⁺ inhibited the bactericidal activities of KCM21 significantly against P. syringae pv. tomato DC3000. The bactericidal kinetic study showed that KCM21 killed both bacteria rapidly and the process was faster against C. michiganensis subsp. michiganensis. The electron microscopic analysis revealed that KCM21 induced the formation of micelles and blebs on the surface of P. syringae pv. tomato DC3000 cells, while it caused cell rupture against C. michiganensis subsp. michiganensis cells. The outer membrane alteration and higher sensitivity to Ca²⁺ suggest that KCM21 interact with the outer membrane of P. syringae pv. tomato DC3000 cells during the process of killing, but not with C. michiganensis subsp. michiganensis cells that lack outer membrane. Considering that both strains had similar sensitivity to KCM21 in LB medium, outer membrane could not be the main target of KCM21, instead common compartments such as cytoplasmic membrane or internal macromolecules might be a possible target(s) of KCM21

Tryptophan-Rich and Proline-Rich Antimicrobial Peptides

Due to the increasing emergence of drug-resistant pathogenic microorganisms, there is a world-wide quest to develop new-generation antibiotics. Antimicrobial peptides (AMPs) are small peptides with a broad spectrum of antibiotic activities against bacteria, fungi, protozoa, viruses and sometimes exhibit cytotoxic activity toward cancer cells. As a part of the native host defense system, most AMPs target the membrane integrity of the microorganism, leading to cell death by lysis. These membrane lytic effects are often toxic to mammalian cells and restrict their systemic application. However, AMPs containing predominantly either tryptophan or proline can kill microorganisms by targeting intracellular pathways and are therefore a promising source of next-generation antibiotics. A minimum length of six amino acids is required for high antimicrobial activity in tryptophan-rich AMPs and the position of these residues also affects their antimicrobial activity. The aromatic side chain of tryptophan is able to rapidly form hydrogen bonds with membrane bilayer components. Proline-rich AMPs interact with the 70S ribosome and disrupt protein synthesis. In addition, they can also target the heat shock protein in target pathogens, and consequently lead to protein misfolding. In this review, we will focus on describing the structures, sources, and mechanisms of action of the aforementioned AMPs

Indirect Force Control of a Cable-Driven Parallel Robot: Tension Estimation using Artificial Neural Network trained by Force Sensor Measurements

In a cable-driven parallel robot (CDPR), force sensors are utilized at each winch motor to measure the cable tension in order to obtain the force distribution at the robot end-effector. However, because of the effects of friction in the pulleys and the unmodeled cable properties of the robot, the measured cable tensions are often inaccurate, which causes force-control difficulties. To overcome this issue, this paper presents an artificial neural network (ANN)-based indirect end-effector force-estimation method, and its application to CDPR force control. The pulley friction and other unmodeled effects are considered as black-box uncertainties, and the tension at the end-effector is estimated by compensating for these uncertainties using an ANN that is developed using the training datasets from CDPR experiments. The estimated cable tensions at the end-effector are used to design a P-controller to track the desired force. The performance of the proposed ANN model is verified through comparisons with the forces measured directly at the end-effector. Furthermore, cable force control is implemented based on the compensated tensions to evaluate the performance of the CDPR in wrench space. The experimental results show that the proposed friction-compensation method is suitable for application in CDPRs to control the cable force

Targeting cancer cells with reactive oxygen and nitrogen species generated by atmospheric-pressure air plasma.

The plasma jet has been proposed as a novel therapeutic method for cancer. Anticancer activity of plasma has been reported to involve mitochondrial dysfunction. However, what constituents generated by plasma is linked to this anticancer process and its mechanism of action remain unclear. Here, we report that the therapeutic effects of air plasma result from generation of reactive oxygen/nitrogen species (ROS/RNS) including H2O2, Ox, OH-, •O2, NOx, leading to depolarization of mitochondrial membrane potential and mitochondrial ROS accumulation. Simultaneously, ROS/RNS activate c-Jun NH2-terminal kinase (JNK) and p38 kinase. As a consequence, treatment with air plasma jets induces apoptotic death in human cervical cancer HeLa cells. Pretreatment of the cells with antioxidants, JNK and p38 inhibitors, or JNK and p38 siRNA abrogates the depolarization of mitochondrial membrane potential and impairs the air plasma-induced apoptotic cell death, suggesting that the ROS/RNS generated by plasma trigger signaling pathways involving JNK and p38 and promote mitochondrial perturbation, leading to apoptosis. Therefore, administration of air plasma may be a feasible strategy to eliminate cancer cells

Treatment of antioxidants reduced level of extra- and intracellular ROS/RNS generated by air plasma.

<p>(a) Levels of extracellular H₂O₂ were determined in culture or non-culture (Medium only) supernatants with air plasma treatment in the presence (AP + NAC) or absence (AP) of the antioxidant NAC (<i>n</i> = 5). NAC was added 1h prior to plasma treatment. The culture supernatant was harvested at the indicated times following air plasma treatment. AP 0 (min) indicates supernatant harvested immediately after plasma treatment. (b) Generation of ROS including H₂O₂, OH⁻, and •O₂ in the intracellular matrix following air plasma jet (AP) were determined using the ROS-sensitive probe H₂DCFDA (<i>n</i> = 5). Cells were pretreated with the antioxidants NAC or cPTIO for 1h and then exposed to air plasma (AP+NAC or AP+cPTIO) or H₂O₂ (H₂O₂+NAC or H₂O₂+cPTIO). At indicated times following air plasma treatment, cells were harvested. The fluorescence of untreated cells (Non-treated) was arbitrarily set to 1. (c) Levels of extracellular NO were determined in non-culture (in the absence of HeLa cells, upper) and culture (in the presence of HeLa cells, bottom) supernatants at the indicated times after air plasma jet exposure by the Griess assay (<i>n</i> = 10). Medium in the presence or absence of HeLa cells was pretreated with NAC or cPTIO for 1h prior to exposure to plasma or H₂O_2. (d) Levels of intracellular NO were evaluated using DAF-FM. Data are shown as the mean ± S.E.M. (<i>n</i> = 10).</p