HaASR2 from Haloxylon ammodendron confers drought and salt tolerance in plants.

Abscisic acid (ABA), stress, and ripening-induced proteins (ASR), which belong to the ABA/WDS domain superfamily, are involved in the plant response to abiotic stresses. Haloxylon ammodendron is a succulent xerohalophyte species that exhibits strong resistance to abiotic stress. In this study, we isolated HaASR2 from H. ammodendron and demonstrated its detailed molecular function for drought and salt stress tolerance. HaASR2 interacted with the HaNHX1 protein, and its expression was significantly up-regulated under osmotic stress. Overexpression of HaASR2 improved drought and salt tolerance by enhancing water use efficiency and photosynthetic capacity in Arabidopsis thaliana. Overexpression of HaASR2 maintained the homeostasis of reactive oxygen species (ROS) and decreased sensitivity to exogenous ABA and endogenous ABA levels by down-regulating ABA biosynthesis genes under drought stress. Furthermore, a transcriptomic comparison between wild-type and HaASR2 transgenic Arabidopsis plants indicated that HaASR2 significantly induced the expression of 896 genes in roots and 406 genes in shoots under osmotic stress. Gene ontology (GO) enrichment analysis showed that those DEGs were mainly involved in ROS scavenging, metal ion homeostasis, response to hormone stimulus, etc. The results demonstrated that HaASR2 from the desert shrub, H. ammodendron, plays a critical role in plant adaptation to drought and salt stress and could be a promising gene for the genetic improvement of crop abiotic stress tolerance.

The Effect of Plasma on Bacteria and Normal Cells in Infected Wound.

Infected wound is one of the most common and serious problem in wound management. Cold atmospheric plasma (CAP) is considered to have a good effect in wound healing as a new type medicine. However, there is a key issue that has not been addressed in the treatment of infected wounds by plasma. Bacteria are always found in the deep region of the wound. When plasma is used to treat wounds, it also acts on normal tissue cells while decontaminating. What is the difference between the same dose of plasma acting on bacteria and normal cells? In this study, the most common bacteria (S. aureus, P. aeruginosa, and E. coli) in infected wound and two kinds of normal skin cells (human keratinocyte and human skin fibroblasts (HSF)) were selected to study the difference of the effects of the same dose of plasma on bacteria and cells. The results reveal that three kinds of 106 CFU mL bacteria could be effectively inactivated by 5 order after plasma treatment 3 min, and P. aeruginosa was more sensitive to plasma (could be inactivated 5 order after 2 min treatment). The 104 mL keratinocyte and HSF were treated with the same dose of plasma; keratinocyte can maintain over 90% of the activity and HSF cells can maintain over 70% of the activity. Moreover, the level of collagen I secreted by HSF increased. Therefore, cells can remain a high activity when a plasma dose capable of inactivating bacteria is applied to them.

Plasma Scalpels: Devices, Diagnostics, and Applications.

The plasma scalpel is an application of gas discharges in electrosurgery. This paper introduces the device structure and physicochemical parameters of the two types of plasma scalpels, namely, a single-electrode Ar discharge device (argon plasma coagulation) and a two-electrode discharge device in normal saline. The diagnostic methods, including the voltage and current characteristics, optical emission spectroscopy, electron spin resonance, and high-speed imaging, are introduced to determine the critical process parameters, such as the plasma power, the gas temperature, the electron density, and the density of active species, and study the ignition dynamics of the plasma discharges in water. The efficacy of the plasma scalpel is mainly based on the physical effects of the electric current and electric field, in addition to the chemical effects of high-density energetic electrons and reactive species. These two effects can be adjusted separately to increase the treatment efficacy of the plasma scalpel. Specific guidance on further improvements of the plasma scalpel devices is also provided.

Effects of Atmospheric Pressure Plasma in the Treatment of Experimental Periodontitis in Beagle Dogs.

OBJECTIVE: The specific objective of this study was to evaluate the effects of atmospheric pressure plasma (APP) in the treatment of experimental periodontitis in Beagle dogs. METHODS: The APP jet was diagnosed using optical emission spectroscopy and laser-induced fluorescence spectroscopy. Six Beagles received stainless steel ligatures to establish experimental periodontitis model. The teeth in the control group were subjected to conventional root surface debridement (RSD) and chlorhexidine irrigation. The APP group also started with RSD and was then subjected to plasma irradiation. Clinical analyses including plaque index, modified sulcus bleeding index, pocket depth and attachment loss (AL), as well as cone-beam computed tomography (CBCT) analysis, were performed at baseline, 4th week, 8th week and 12th week after treatment. RESULTS: The results showed that typical reactive oxygen and nitrogen species were found in the full spectrum and the gas temperature of APP was close to room temperature. The highest concentrations of hydroxide and oxygen were obtained at 5 mm away from the nozzle. In both groups, all values in clinical examinations were significantly lower (P<0.05) at 12th week after treatment than those at baseline. At the 12th week, the AL in clinical examinations and the bone loss in CBCT images in the APP group were significantly lower than those in the control group (P<0.05). The hematoxylin-eosin staining showed more renascent alveolar bone in the APP group than in the control group. CONCLUSION: These findings suggested that APP has profound potential for use as an adjunct approach for periodontitis treatment.

Matrix Metalloproteinase-1 (MMP1) Upregulation through Promoter Hypomethylation Enhances Tamoxifen Resistance in Breast Cancer.

BACKGROUND: Tamoxifen (tam) is widely used to treat estrogen-positive breast cancer. However, cancer recurrence after chemotherapy remains a major obstacle to achieve good patient prognoses. In this study, we aimed to identify genes responsible for epigenetic regulation of tam resistance in breast cancer. METHODS: Methylation microarray data were analyzed to screen highly hypomethylated genes in tam resistant (tamR) breast cancer cells. Quantitative RT-PCR, Western blot analysis, and immunohistochemical staining were used to quantify expression levels of genes in cultured cells and cancer tissues. Effects of matrix metalloproteinase-1 (MMP1) expression on cancer cell growth and drug resistance were examined through colony formation assays and flow cytometry. Xenografted mice were generated to investigate the effects of MMP1 on drug resistance in vivo. RESULTS: MMP1 was found to be hypomethylated and overexpressed in tamR MCF-7 (MCF-7/tamR) cells and in tamR breast cancer tissues. Methylation was found to be inversely associated with MMP1 expression level in breast cancer tissues, and patients with lower MMP1 expression exhibited a better prognosis for survival. Downregulating MMP1 using shRNA induced tam sensitivity in MCF-7/tamR cells along with increased apoptosis. The xenografted MCF-7/tamR cells that stably expressed short hairpin RNA (shRNA) against MMP1 exhibited retarded tumor growth compared to that in cells expressing the control shRNA, which was further suppressed by tam. CONCLUSIONS: MMP1 can be upregulated through promoter hypomethylation in tamR breast cancer, functioning as a resistance driver gene. MMP1 can be a potential target to suppress tamR to achieve better prognoses of breast cancer patients.

In vitro and in vivo research of atmosphere pressure nonequilibrium plasmas on root canal disinfection: implication for alternative strategy for irrigation.

OBJECTIVE: To investigate an intracanal disinfection methodology of APNPs (atmosphere pressure nonequilibrium plasmas) or modified APNPs in root canal treatment and evaluate the antimicrobial efficiency against in vitro infected dentinal tubules and in vivo experimental apical periodontitis. MATERIALS AND METHODS: Dentine specimens were centrifugated with Enterococcus faecalis to generate 1-day-old and 3-week-old biofilms, and were treated with 2% chlorhexidine (Chx), APNP or modified APNP for 3 and 10 min (n=4). LIVE/DEAD staining was employed to analyze the ratio of deactivated bacteria. Experimental apical periodontitis in beagles was induced. Root canal therapy with APNPs or modified APNPs was performed and the antimicrobial effect was evaluated by histological and radiographical analyses. RESULTS: APNP deactivated 1-day-old and 3-week-old E. feacalis in dentinal tubules as much as 2% Chx irrigating. Modified APNP significantly deactivated more E. faecalis biofilms in dentinal tubules for 3-min and 10-min treatments, without thermal damage or dentinal destruction being observed. In beagles' apical periodontitis, significantly increased BV/TV and decreased lesion volume of apical bone were found in modified APNP group than 2% Chx irrigation group according to µCT. Fewer inflammatory cells and bacterial residual in dentine were observed in modified APNP-treated apical tissue by histology staining compared with those in the 2% Chx irrigation group. CONCLUSION: The antimicrobial effect of APNP jet irradiation was comparable to that of 2% Chx irrigation. No structural damage in dentine or tissue necrosis at the periapical region was induced upon treatment. The modified APNP demonstrated an increased antimicrobial efficacy compared with 2% Chx irrigation both in vitro and in vivo. CLINICAL RELEVANCE: The modified APNPs can be used as an alternative intracanal disinfection strategy.

Effects of Cold Jet Atmospheric Pressure Plasma on the Structural Characteristics and Immunoreactivity of Celiac-Toxic Peptides and Wheat Storage Proteins.

The present research reported the effects of structural properties and immunoreactivity of celiac-toxic peptides and wheat storage proteins modified by cold jet atmospheric pressure (CJAP) plasma. It could generate numerous high-energy excited atoms, photons, electrons, and reactive oxygen and nitrogen species, including O3, H2O2, •OH, NO2- and NO3- etc., to modify two model peptides and wheat storage proteins. The Orbitrap HR-LC-MS/MS was utilized to identify and quantify CJAP plasma-modified model peptide products. Backbone cleavage of QQPFP and PQPQLPY at specific proline and glutamine residues, accompanied by hydroxylation at the aromatic ring of phenylalanine and tyrosine residues, contributed to the reduction and modification of celiac-toxic peptides. Apart from fragmentation, oxidation, and agglomeration states were evaluated, including carbonyl formation and the decline of γ-gliadin. The immunoreactivity of gliadin extract declined over time, demonstrating a significant decrease by 51.95% after 60 min of CJAP plasma treatment in vitro. The CJAP plasma could initiate depolymerization of gluten polymer, thereby reducing the amounts of large-sized polymers. In conclusion, CJAP plasma could be employed as a potential technique in the modification and reduction of celiac-toxic peptides and wheat storage proteins.

Microplasma Bubbles: Reactive Vehicles for Biofilm Dispersal.

Interactions between effects generated by cold atmospheric-pressure plasmas and water have been widely investigated for water purification, chemical and nanomaterial synthesis, and, more recently, medicine and biotechnology. Reactive oxygen and nitrogen species (RONS) play critical roles in transferring the reactivity from gas plasmas to solutions to induce specific biochemical responses in living targets, e.g., pathogen inactivation and biofilm removal. While this approach works well in a single-organism system at a laboratory scale, integration of plasma-enabled biofilm removal into complex real-life systems, e.g., large aquaculture tanks, is far from trivial. This is because it is difficult to deliver sufficient concentrations of the right kind of species to biofilm-covered surfaces while carefully maintaining a suitable physiochemical environment that is healthy for its inhabitants, e.g., fish. In this work, we show that underwater microplasma bubbles (generated by a microplasma-bubble reactor that forms a dielectric barrier discharge at the gas-liquid interface with the applied voltage of 4.0 kV) act as transport vehicles to efficiently deliver reactive plasma species to the target biofilm sites on artificial and living surfaces while keeping healthy water conditions in a multispecies system. The as-generated air microplasma bubbles and plasma-activated water (PAW) both can effectively reduce the existing pathogenic biofilm load by ∼83 and 60%, respectively, after 15 min of discharge at 40 W and prevent any new biofilm from forming. The generation of underwater microplasma bubbles in a custom-made fish tank for less than a minute per day (20 s per time, twice daily) can introduce sufficient quantities of RONS into PAW to reduce the biofilm-infected area by ∼80-90% and improve the health status of Cichlasoma synspilum × Cichlasoma citrinellum blood parrot cichlid fish. Species generated include hydrogen peroxide, ozone, nitrite, nitrate, and nitric oxide. Using mimicked chemical solutions, we show that the plasma-induced nitric oxide acts as a critical bioactive species that triggers the release of cells from the biofilm and their inactivation.

Dynamic responses of Haloxylon ammodendron to various degrees of simulated drought stress.

Haloxylon ammodendron, a C4 perennial, succulent and xero-halophytic shrub, is highly resistant to harsh environments, therefore, exploring the stress resistance mechanism will be beneficial for the use of xerophytes to prevent desertification. To determine osmotic adjustment (OA) and antioxidase functions under simulated drought stress, 8-week-old seedlings were treated with sorbitol solutions to maintain osmotic potentials (Ψs) at a control and -0.5 and -1.0 MPa. Under -0.5 MPa osmotic stress, H. ammodendron stably maintained the water content of assimilating branches, a result that was not significantly different from the result of the control group. Moreover, the Ψs decreased significantly, which helped plants absorb water efficiently from the environment, as H. ammodendron accumulated massive osmotic regulators in its assimilating branches to adjust shoot Ψs. Specifically, the contribution of Na+ to shoot Ψs was up to 45%, and Na+ became the main osmotic regulator of OA. During the treatments, the content and contribution of K+ remained stable. However, the total contribution of three organic osmotic regulators (free proline, betaine and soluble sugar) was only 20%, and betaine was the main organic osmotic regulator, accounting for approximately 15% of the 20% contribution. Moreover, H. ammodendron seedlings presented strong antioxidases, especially when there was a high activity level of superoxide dismutase, and with an increase in treatment time and degree of osmotic stress, the activity of peroxidase and catalase increased significantly. Substantial accumulation of osmotic adjustment substances was an important strategy for H. ammodendron to cope with simulated drought stress, in particular, H. ammodendron absorbed much Na+ and transported Na+ into the assimilating branch for OA. The scavenging of reactive oxygen species by antioxidases was another adaptation strategy for H. ammodendron to adapt to simulated drought stress.

Cold atmospheric plasma ameliorates imiquimod-induced psoriasiform dermatitis in mice by mediating antiproliferative effects.

Psoriasis is a chronic hyperproliferative skin disease characterised by excessive growth of keratinocytes. Indeed, inducing keratinocyte apoptosis is a key mechanism responsible for psoriatic plaques clearance following some important existing therapies, which display pro-oxidant activity. Cold atmospheric plasma (CAP), acting as a tuneable source of reactive oxygen and nitrogen species (RONS), can controllably transfer RONS to the cellular environment, deliver antiproliferative RONS concentrations and exert antiproliferative and proapoptotic effects. This study was undertaken to evaluate the therapeutic potential of CAP in psoriasis. We used cell models of psoriasis-like inflammation by adding lipopolysaccharide (LPS) or tumour necrosis factor alpha (TNF-α) to HaCaT keratinocytes. Indirect plasma, plasma-activated medium (PAM), was administered to HaCaT cells. Atmospheric pressure plasma jet (APPJ) was applied directly to imiquimod (IMQ)-induced psoriasiform dermatitis in mice. The results showed that PAM induced an increase in intracellular ROS and caused keratinocyte apoptosis. Moreover, cells under inflammation showed lesser viability and larger apoptosis rate. With repeated administration of APPJ, psoriasiform lesions showed ameliorated morphological manifestation and reduced epidermal proliferation. Overall, this study supports that CAP holds good potential in psoriasis treatment.

Plasma Activated Oil: Fast Production, Reactivity, Stability, and Wound Healing Application.

The "third-generation" cooking oil based drug, named plasma activated oil (PAO), is produced in cheap olive oil by a single-step, room temperature, energy-efficient, and environment-friendly dry plasma-enabled process. The streamer-surface discharge generates abundant energetic species, atomic oxygen at the plasma-oil interface. The otherwise challenging dissociation of C═C double bonds by energetic species and oxidation by the plasma generated atomic oxygen is the key mechanism to produce the H2O2 active species and carboxylic acid in the PAO. It is shown that the peroxide value and acid value of PAO are 7.5 times and 57% higher than those of the traditional ozonated oil, respectively. Different from plasma activated water whose shelf life was less than 1 week, PAO could be stored at room temperature for at least 3 months, and a shelf life of up to 1 year is expected. We further reveal that the PAO can not only sterilize the wound, but also promote more release of growth factor such as VEGF and CD34; therefore, the wound healing of PAO is 28.5% faster than that of the control group.

Medical applications of nonthermal atmospheric pressure plasma in dermatology.

Plasma is an ionized gas that consists of positively and negatively charged particles, neutral atoms, and photons. Recent developments in plasma sources have made it possible to generate room-temperature plasma in the "open air", thus enabling the application of plasma in vivo. Using nonthermal plasma, active agents can be efficiently delivered to target cells without creating thermal damage. Also known as cold atmospheric pressure plasma (CAP), nonthermal atmospheric pressure plasma offers innovative medical applications. In this context, it has also gained wide attention in the field of dermatology. The complex and variable mixture of active agents in plasma - predominantly reactive oxygen and nitrogen species (ROS, RNS) - can control or trigger complex biochemical reactions, achieving the desired effects in a dose-dependent manner. The objective of the present review is to present potential applications of plasma in dermatology and analyze its potential mechanisms of action.

Transcriptomic Profiling and Physiological Analysis of Haloxylon ammodendron in Response to Osmotic Stress.

Haloxylon ammodendron, a perennial xero-halophyte, is an essential species for investigating the effects of drought on desert tree. To gain a comprehensive knowledge on the responses of H. ammodendron to drought stress, we specially performed the molecular and physiological analysis of H. ammodendron in response to -0.75 MPa osmotic stress for six and 24 h in lab condition via RNA-seq and digital gene expression (DGE). In total, 87,109 unigenes with a mean length of 680 bp and 13,486 potential simple sequence repeats (SSRs) were generated, and 3353 differentially expressed genes (DEGs) in shoots and 4564 in roots were identified under stress. These DEGs were mainly related to ion transporters, signal transduction, ROS-scavenging, photosynthesis, cell wall organization, membrane stabilization and hormones. Moreover, the physiological changes of inorganic ions and organic solute content, peroxidase (POD) activity and osmotic potential were in accordance with dynamic transcript profiles of the relevant genes. In this study, a detailed investigation of the pathways and candidate genes identified promote the research on the molecular mechanisms of abiotic stress tolerance in the xero-halophytic species. Our data provides valuable genetic resources for future improvement of forage and crop species for better adaptation to abiotic stresses.

Bactericidal effect of plasma jet with helium flowing through 3% hydrogen peroxide against Enterococcus faecalis.

The aim of the present study was to assess the antimicrobial activity of plasma jet with helium (He) flowing through 3% hydrogen peroxide in root canals infected with Enterococcus faecalis. A total of 42 single-rooted anterior teeth were prepared, sterilized, inoculated with an E. faecalis suspension and incubated for 7 days. Next, the teeth were randomly divided into six experimental groups (including groups treated by plasma jet with or without He for different time durations) and one control group treated without plasma. The number of surviving bacteria in each canal was determined by counting the colony forming units (CFU)/ml on nutrient agar plates. The results indicated that statistically significant reduction in CFU/ml (P<0.005) existed for all treatment groups relative to the control group. The greatest reductions in CFU/ml were observed for Group 3 (7.027 log unit reduction) and Group 2 (6.237 log unit reduction), which were treated by plasma jet sterilization with He flowing through 3% hydrogen peroxide for 4 min or for 2 min, respectively. In addition, the reduction in Group 3 was significantly greater compared with that in Group 2 or in the groups treated by plasma jet sterilization without He flowing through 3% hydrogen peroxide for 1, 2 or 4 min. In conclusion, plasma jet with or without He flowing through 3% hydrogen peroxide can effectively sterilized root canals infected with E. faecalis and should be considered as an alternative method for root canal disinfection in endodontic treatments.

Cold plasma welding of polyaniline nanofibers with enhanced electrical and mechanical properties.

Joining conducting polymer (CP) nanofibers into an interconnected porous network can result in good mechanical and electrical contacts between nanofibers that can be beneficial for the high performance of CP-based devices. Here, we demonstrate the cold welding of polyaniline (PAni) nanofiber loose ends with cold plasma. The room-temperature and atmospheric-pressure helium micro-plasma jet launches highly charged ion bullets at a PAni nanofiber target with high precision and the highly charged ion bullet selectively induces field emission at the sharp nanofiber loose ends. This technique joins nanofiber tips without altering the morphology of the film and protonation thus leading to significantly enhanced electrical and mechanical properties. In addition, this technique has high spatial resolution and is able to selectively weld and dope regions of nanofiber film with promising novel device applications.

Effects of non-equilibrium plasma in the treatment of ligature-induced peri-implantitis.

AIM: To evaluate the effects of non-equilibrium plasma in the treatment of ligature-induced peri-implantitis in beagle dogs. MATERIALS AND METHODS: Six beagles received 12 implants installed in the position of the fourth mandibular premolars. Ligature-induced peri-implantitis was initiated at 3 months post-implantation. When approximately 40% of the supporting bone was lost, the ligatures were removed. The implants were subjected to the muco-periosteal scaling and chlorhexidine irrigation with or without plasma irrigation. Three months later, clinical, radiographic and microbiological analyses were performed. Block biopsies were prepared for micro-CT and histomorphometric analysis. The primary outcome was the difference in bone healing of peri-implant sites, and the secondary outcomes included changes in clinical parameters (SBI, PD) and bacterial detection. RESULTS: At baseline, no significant differences were observed between the two groups. At 3 months post-treatment, the plasma group showed a significantly higher bone level than the control group (p < 0.05), a significantly decreased detection of bacteria (Porphyromonas gingivalis and Tannerella forsythia) (p < 0.05), and a significant improvement in clinical examination (p < 0.05). CONCLUSIONS: Within the limits of this study, non-equilibrium plasma treatment as an adjunct to the conventional therapy is a feasible approach for the treatment of peri-implantitis.

Beneficial soil bacterium Bacillus subtilis (GB03) augments salt tolerance of white clover.

Soil salinity is an increasingly serious problem worldwide that reduces agricultural output potential. Selected beneficial soil bacteria can promote plant growth and augment tolerance to biotic and abiotic stresses. Bacillus subtilis strain GB03 has been shown to confer growth promotion and abiotic stress tolerance in the model plant Arabidopsis thaliana. Here we examined the effect of this beneficial soil bacterium on salt tolerance in the legume forage crop, white clover. Plants of white clover (Trifolium repens L. cultivar Huia) were grown from seeds with or without soil inoculation of the beneficial soil bacterium Bacillus subtilis GB03 supplemented with 0, 50, 100, or 150 mM NaCl water into soil. Growth parameters, chlorophyll content, malondialdehyde (MDA) content and osmotic potential were monitored during the growth cycle. Endogenous Na(+) and K(+) contents were determined at the time of harvest. White clover plants grown in GB03-inoculated soil were significantly larger than non-inoculated controls with respect to shoot height, root length, plant biomass, leaf area and chlorophyll content; leaf MDA content under saline condition and leaf osmotic potential under severe salinity condition (150 mM NaCl) were significantly decreased. Furthermore, GB03 significantly decreased shoot and root Na(+) accumulation and thereby improved K(+)/Na(+) ratio when GB03-inoculated plants were grown under elevated salt conditions. The results indicate that soil inoculation with GB03 promotes white clover growth under both non-saline and saline conditions by directly or indirectly regulating plant chlorophyll content, leaf osmotic potential, cell membrane integrity and ion accumulation.

Single-cell-precision microplasma-induced cancer cell apoptosis.

The issue of single-cell control has recently attracted enormous interest. However, in spite of the presently achievable intracellular-level physiological probing through bio-photonics, nano-probe-based, and some other techniques, the issue of inducing selective, single-cell-precision apoptosis, without affecting neighbouring cells remains essentially open. Here we resolve this issue and report on the effective single-cell-precision cancer cell treatment using the reactive chemistry of the localized corona-type plasma discharge around a needle-like electrode with the spot size ∼1 µm. When the electrode is positioned with the micrometer precision against a selected cell, a focused and highly-localized micro-plasma discharge induces apoptosis in the selected individual HepG2 and HeLa cancer cells only, without affecting any surrounding cells, even in small cell clusters. This is confirmed by the real-time monitoring of the morphological and structural changes at the cellular and cell nucleus levels after the plasma exposure.

Effect of modified nonequilibrium plasma with chlorhexidine digluconate against endodontic biofilms in vitro.

INTRODUCTION: Nonequilibrium plasma has been reported to effectively kill Enterococcus faecalis in endodontic biofilm compared with chlorhexidine digluconate (CHX). The purpose of this study was to evaluate the antimicrobial in vitro activity of modified nonequilibrium plasma with CHX against E. faecalis and multispecies biofilms on bovine dentin discs. METHODS: Sterile bovine dentin discs were incubated with E. faecalis or a mixture of bacteria from human dental root canal infections to form 1- and 3-week-old biofilms. The specimens were subjected to nonequilibrium plasma, modified nonequilibrium plasma with CHX, and 2% CHX for 2- and 5-minute exposure. After treatment, the biofilms were stained with viability dyes and examined by confocal laser scanning microscopy and 3-dimensional reconstruction analysis. The proportions of bacterial cells killed by the treatments were calculated. RESULTS: The 3-dimensional reconstruction images showed that 1- and 3-week-old biofilms adhered to bovine dentin discs. The proportions of dead cells increased significantly with the longer exposure in each treatment group (P < .05). Modified nonequilibrium plasma was the most effective in killing bacteria in E. faecalis and multispecies biofilms at both 2 and 5 minutes (P < .05). No significant difference was detected between nonequilibrium plasma and CHX groups (P > .05). Significantly more cells were killed in 1-week-old biofilms than in 3-week-old biofilms in all groups (P < .05). CONCLUSIONS: The modified nonequilibrium plasma killed more bacteria than conventional nonequilibrium plasma and 2% CHX in E. faecalis and multispecies endodontic biofilms in vitro and thus shows promise as an additional tool in infection control during endodontic treatment.