Synergistic effects of atmospheric pressure plasma‐emitted components on DNA oligomers: a Raman spectroscopic study

Cold atmospheric‐pressure plasmas have become of increasing importance in sterilization processes especially with the growing prevalence of multi‐resistant bacteria. Albeit the potential for technological application is obvious, much less is known about the molecular mechanisms underlying bacterial inactivation. X‐jet technology separates plasma‐generated reactive particles and photons, thus allowing the investigation of their individual and joint effects on DNA. Raman spectroscopy shows that particles and photons cause different modifications in DNA single and double strands. The treatment with the combination of particles and photons does not only result in cumulative, but in synergistic effects. Profilometry confirms that etching is a minor contributor to the observed DNA damage in vitro.

Schematics of DNA oligomer treatment with cold atmospheric‐pressure plasma.     

Bactericidal efficacy of tissue tolerable plasma on microrough titanium dental implants: An in‐vitro‐study

Surface decontamination remains challenging in peri‐implant infection therapy. To investigate the bactericidal efficacy of tissue tolerable plasma, S. mitis biofilms were created in vitro on 32 microrough titanium dental implants. Biofilm imaging was performed by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). The implants were either rinsed with 1% NaCl as negative control (C) or irradiated with a diode laser (DL) for 60 sec as positive control or plasma (TTP60, TTP120) for 60 or 120 sec. Subsequently, colony forming units (CFU) were counted. Post‐treatment, implants were further examined using fluorescence microscopy (FM). Median CFU counts differed significantly between TTP60, TTP120 and C (2.19 and 2.2 vs. 3.29 log CFU/ml; p = 0.012 and 0.024). No significant difference was found between TTP60 and TTP120 (p = 0.958). Logarithmic reduction factors were (TTP60) 2.21, (TTP120) 1.93 and (DL) 0.59. Prior to treatment, CLSM and SEM detected adhering bacteria. Post‐treatment FM recorded that the number of dead cells was higher using TTP compared to DL and C. In view of TTP's effectiveness, regardless of resistance patterns and absence of surface alteration, its use in peri‐implant infection therapy is promising. The results encourage conducting clinical studies to investigate its impact on relevant parameters.

Experimental Design     

Antimicrobial mechanism and the effect of atmospheric pressure N2 plasma jet on the regeneration capacity of Staphylococcus aureus biofilm

Abstract

This study systematically assessed the inactivation mechanism on Staphylococcus aureus biofilms by a N₂ atmospheric-pressure plasma jet and the effect on the biofilm regeneration capacity from the bacteria which survived, and their progenies. The total bacterial populations were 7.18?±?0.34 log10 CFU ml^?1 in biofilms and these were effectively inactivated (>5.5-log10 CFU ml^?1) within 30?min of exposure. Meanwhile, >80% of the S. aureus biofilm cells lost their metabolic capacity. In comparison, ～20% of the plasma-treated bacteria entered a viable but non-culturable state. Moreover, the percentage of membrane-intact bacteria declined to ～30%. Scanning electron microscope images demonstrated cell shrinkage and deformation post-treatment. The total amount of intracellular reactive oxygen species was observed to have significantly increased in membrane-intact bacterial cells with increasing plasma dose. Notably, the N₂ plasma treatment could effectively inhibit the biofilm regeneration ability of the bacteria which survived, leading to a long-term phenotypic response and dose-dependent inactivation effect on S. aureus biofilms, in addition to the direct rapid bactericidal effect.     

Treatment of Streptococcus mutans biofilms with a nonthermal atmospheric plasma

AIMS: A nonthermal atmospheric plasma, designed for biomedical applications, was tested for its antimicrobial activity against biofilm cultures of a key cariogenic bacterium Streptococcus mutans. METHODS AND RESULTS: The Strep. mutans biofilms were grown with and without 0.15% sucrose. A chlorhexidine digluconate rinse (0.2%) was used as a positive antimicrobial reference. The presence of sucrose and the frequency of plasma application during growth were shown to have a significant effect on the response to treatment and antibacterial activity. CONCLUSIONS: A single plasma treatment for 1 min on biofilms cultured without sucrose caused no re-growth within the observation period. However, with either single or repeated plasma treatments of 1 min, on biofilms cultured with 0.15% sucrose, growth was only reduced. SIGNIFICANCE AND IMPACT OF THE STUDY: In summary, there may be a role for nonthermal plasma therapies in dental procedures. Sucrose and associated growth conditions may be a factor in the survival of oral biofilms after treatment.     

Novel mutation breeding method for Streptomyces avermitilis using an atmospheric pressure glow discharge plasma

Aims: Avermectins are major antiparasitic agents used commercially in animal health, agriculture and human infections. To improve the fermentation efficiency of avermectins, for the first time a plasma jet generated by a novel atmospheric pressure glow discharge (APGD) was employed to generate mutations in Streptomyces avermitilis. Methods and Results: The APGD plasma jet, driven by a radio frequency (RF) power supply with water‐cooled and bare‐metallic electrodes, was used as a new mutation method to treat the spores of S. avermitilis. The plasma jet yielded high total (over 30%) and positive (about 21%) mutation rates on S. avermitilis, and a mutated strain, designated as G1‐1 with high productivity of avermectin B1a and genetic stability, was obtained. Conclusions: Because of the low jet temperature, the high concentrations of the chemically reactive species and the flexibility of its operation, the RF APGD plasma jet has a strong mutagenic effect on S. avermitilis. Significance and Impact of the Study: This is a proof‐of‐concept study for the use of an RF APGD plasma jet for inducing mutations in microbes. We have shown that the RF APGD plasma jet could be developed as a promising and convenient mutation tool for the fermentation industry and for use in biotechnology research.     

等离子体射流灭活液体中铜绿假单胞菌的研究

【目的】测定等离子射流对铜绿假单胞菌(Pseudomonas aeruginosa)的灭活效果,探究低温等离子体射流的杀菌机理。【方法】采用平板计数法测定等离子体射流的杀菌效果,荧光显微镜、透射电镜观察等离子体作用后菌体结构的变化,蛋白浓度测定和SDS-PAGE电泳检测菌液上清液中可溶性蛋白的泄漏量。【结果】等离子体射流处理铜绿假单胞菌菌液5 min,杀灭率可达到99.9%以上。透射电镜观察可见细菌菌体结构发生改变,细胞壁、细胞膜损伤破裂,细胞内容物泄露。进一步对处理铜绿假单胞菌上清液中的蛋白质含量变化进行检测,结果显示随着处理时间的增加,上清液中蛋白质含量持续增加,在2 min时达到最大值。【结论】等离子体射流可以通过破坏细胞结构造成细胞质泄露,使其丧失正常的细胞功能,从而达到快速有效地杀灭铜绿假单胞菌的效果。     

Surface molecules on HaCaT keratinocytes after interaction with non‐thermal atmospheric pressure plasma

Non‐thermal atmospheric‐pressure plasmas have been developed that will be used in future for several purposes, e.g. medicine. Living tissues and cells are at the focus of plasma treatment, e.g. to improve wound healing, or induce apoptosis and growth arrest in tumour cells. Detailed investigations of plasma‐cell interactions are needed. Cell surface adhesion molecules as integrins, cadherins or the EGFR (epidermal growth factor receptor) are of importance in wound healing and also for development of cancer metastasis. This study has focused on measurement of cell surface molecules on human HaCaT keratinocytes (human adult low calcium temperature keratinocytes) promoting adhesion, migration and proliferation as one important feature of plasma‐cell interactions. HaCaT keratinocytes were treated with plasma by a surface dielectric barrier discharge in air. Cell surface molecules and induction of intracellular ROS (reactive oxygen species) were analysed by flow cytometry 24 h after plasma treatment. Besides a reduction of cell viability a significant down‐regulation of E‐cadherin and the EGFR expression occurred. The influence on α₂‐ and β₁‐integrins was less pronounced, and expression of ICAM‐1 (intercellular adhesion molecule 1) was unaffected. The extent of effects depended on the exposure time of cells to the plasma and the treatment regimen. Intracellular level of ROS detected by the fluorescent dye H₂DCFDA (2′,7′‐dichlorodihydrofluorescein diacetate) increased by plasma treatment, but it was neither dependent on the treatment time nor related to the different treatment regimens. Two‐dimensional cultures of HaCaT keratinocytes appear to be a suitable method of investigating plasma‐cell interactions.     

Cold atmospheric plasma induces apoptosis of melanoma cells via Sestrin2‐mediated nitric oxide synthase signaling

Cold atmospheric plasma (CAP) represents a promising therapy for selectively cancer killing. However, the mechanism of CAP‐induced cancer cell death remains unclear. Here, we identified the tumor necrosis factor‐family members, especially Fas, and overloaded intracellular nitric oxide participated in CAP induced apoptosis in A375 and A875 melanoma cell lines, which was known as extrinsic apoptosis pathway. This progress was mediated by antagonistic protein of reactive oxygen species, Sestrin2. The over expression of Sestrin2 induced by plasma treatment resulted in phosphorylation of p38 mitogen‐activated protein kinase (MAPK), followed by increased expression of nitric oxide synthase (iNOS), Fas and Fas ligand. Depletion of Sestrin2 reduced iNOS and Fas expression, which was associated with reduction of plasma‐induced apoptosis. In contrast, inhibition of iNOS activity and phosphorylation of p38 did not alter Sestrin2 expression in plasma‐treated melanoma cells. Taken together, cold atmospheric plasma increases Sestrin2 expression and further activates downstream iNOS, Fas and p38 MAPK signaling to induce apoptosis of melanoma cell lines. These findings suggest a previously unrecognized mechanism in melanoma cells response to cold atmospheric plasma therapy.      

ARTP诱变猴头菌株的发酵菌丝体多糖理化性质及体外免疫活性

为探讨常压室温等离子体诱变的3株高产多糖猴头菌和出发菌株的多糖组分差异,通过液体发酵获得的菌丝体经水提、分级醇沉获得8个胞内多糖组分,对它们的理化性质、结构特征及体外免疫活性进行了研究。结果表明,3株ARTP诱变菌株414、321、236菌丝体多糖含量较出发菌株有较明显提升;ARTP诱变的猴头菌20%醇沉多糖组分较出发菌株分子量大,所占比例增加;诱变菌株60%醇沉多糖组分的分子量略大于出发菌株,所占比例相近。20%醇沉多糖主要由半乳糖、葡萄糖、甘露糖构成,诱变菌株该多糖组分中葡萄糖和甘露糖的比例较出发菌株均有明显提升,60%醇沉多糖组分单糖组成无明显差异;8个多糖组分均具有体外刺激巨噬细胞释放NO的活性,其中20%醇沉多糖的活性优于60%醇沉多糖,诱变菌株的生物活性优于出发菌株。本研究探讨了ARTP诱变对猴头菌胞内多糖结构及活性的影响,为猴头菌相关产品的开发提供了优质资源。     

Effect of pressure on helix‐coil transition of an alanine‐based peptide: An FTIR study

Protein–protein interactions are mediated by complementary amino acids defining complementary surfaces. Typically not all members of a family of related proteins interact equally well with all members of a partner family; thus analysis of the sequence record can reveal the complementary amino acid partners that confer interaction specificity. This article develops methods for learning and using probabilistic graphical models of such residue “cross‐coupling” constraints between interacting protein families, based on multiple sequence alignments and information about which pairs of proteins are known to interact. Our models generalize traditional consensus sequence binding motifs, and provide a probabilistic semantics enabling sound evaluation of the plausibility of new possible interactions. Furthermore, predictions made by the models can be explained in terms of the underlying residue interactions. Our approach supports different levels of prior knowledge regarding interactions, including both one‐to‐one (e.g., pairs of proteins from the same organism) and many‐to‐many (e.g., experimentally identified interactions), and we present a technique to account for possible bias in the represented interactions. We apply our approach in studies of PDZ domains and their ligands, fundamental building blocks in a number of protein assemblies. Our algorithms are able to identify biologically interesting cross‐coupling constraints, to successfully identify known interactions, and to make explainable predictions about novel interactions. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Large and small forms of cholecystokinin in human plasma: measurement using high pressure liquid chromatography and radioimmunoassay

Defining the role of cholecystokinin (CCK) in gut physiology and disease has proved difficult because of problems with development of radioimmunoassays and because CCK exists in several different molecular forms which have different biological actions. In order to measure small (8 amino acid residues, CCK 8) and large (33 and 39 residues) forms of CCK in plasma we have developed high pressure liquid chromatographic (HPLC) fractionation of plasma prior to radioimmunoassay. Fasting plasma CCK 8 and CCK $\text{33}\text{39}$ levels were usually undetectable (< 3 and < 6 pmol/1, respectively). After a liquid fat meal both CCK 8 and CCK $\text{33}\text{39}$ levels were significantly elevated at 5 min (11.3±3.3 and 11.6±2.6 pmol/1, respectively). Peak CCK 8 levels occurred at 30 min (15.0±4.4 pmol/1) while peak CCK $\text{33}\text{39}$ levels occurred at 120 min (16.7±4.9 pmol/1. Total CCK levels showed a biphasic response to the meal. These CCK 8 and CCK $\text{33}\text{39}$ responses to oral fat are consistent with a role for these hormones in the regulation of gallbladder emptying and pancreatic secretion.     

On the production of glycogen by Pseudomonas fluorescens during biofilm development: an in situ study by attenuated total reflection-infrared with chemometrics

Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy was used to monitor Pseudomonas fluorescens biofilms in situ, non-destructively, in real time, and under fully hydrated conditions. Changes accompanying the metabolic evolution of the sessile bacterial cells from the nascent biofilm monolayer to the beginning of the multi-layered structure in the presence of nutrients were identified via the ATR-FTIR fingerprints of the young biofilm on the ATR crystal. The ATR-FTIR spectra were analysed by classical methods (time evolution of integrated intensities and profile evolution of specific bands), and also by a multivariate curve resolution, Bayesian positive source separation, to extract the pure component spectra and their change of concentration over time occurring during biofilm settlement. This work showed clearly the overproduction of glycogen by sessile P. fluorescens, which had not previously been described by other research groups.     

Resettlement challenges and dilemmas: An in‐depth case study of Bhutanese refugee women in Australia

In this paper, the perceptions of Bhutanese refugee women were explored in relation to their changing identity and their behavioural responses in the use of preventive health services following resettlement. Interviews with 30 Bhutanese refugee women in Melbourne revealed resettlement drivers, challenges and dilemmas. There was no option for women other than resettlement, women wanted to escape from bad conditions and hopes for a better future for their children was critical in their thinking. Resettlement introduced new unexpected challenges including cultural discontinuity, changed dependency and a sense of not belonging. Dilemmas were apparent in their experiences including: despondency and contentment, gratitude and regret; and identifying as either Australian or refugee. Motherhood was important to women in this study and was challenged by changed dependency with their children in resettlement. Challenges of cultural discontinuity included the impact of communication difficulties and illiteracy and the absence of social connections with neighbours. A better understanding of how changing identities of women interact should lead to more effective strategies including tailored programs and activities.     

Synthetic antimicrobial β‐peptide in dual‐treatment with fluconazole or ketoconazole enhances the in vitro inhibition of planktonic and biofilm Candida albicans

Fungal infections are a pressing concern for human health worldwide, particularly for immunocompromised individuals. Current challenges such as the elevated toxicity of common antifungal drugs and the emerging resistance towards these could be overcome by multidrug therapy. Natural antimicrobial peptides, AMPs, in combination with other antifungal agents are a promising avenue to address the prevailing challenges. However, they possess limited biostability and susceptibility to proteases, which has significantly hampered their development as antifungal therapies. β‐peptides are synthetic materials designed to mimic AMPs while allowing high tunability and increased biostability. In this work, we report for the first time the inhibition achieved in Candida albicans when treated with a mixture of a β‐peptide model and fluconazole or ketoconazole. This combination treatment enhanced the biological activity of these azoles in planktonic and biofilm Candida, and also in a fluconazole‐resistant strain. Furthermore, the in vitro cytotoxicity of the dual treatment was evaluated towards the human hepatoma cell line, HepG2, a widely used model derived from liver tissue, which is primarily affected by azoles. Analyses based on the LA‐based method and the mass‐action law principle, using a microtiter checkerboard approach, revealed synergism of the combination treatment in the inhibition of planktonic C. albicans. The dual treatment proved to be fungicidal at 48 and 72 h. Interestingly, it was also found that the viability of HepG2 was not significantly affected by the dual treatments. Finally, a remarkable enhancement in the inhibition of the highly azole‐resistant biofilms and fluconazole resistant C. albicans strain was obtained. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.