Abstract

Biofilm-associated chronic Pseudomonas aeruginosa lung infections in patients with cystic fibrosis are virtually impossible to eradicate with antibiotics because biofilm-growing bacteria are highly tolerant to antibiotics and host defense mechanisms. Previously, we found that ginseng treatments protected animal models from developing chronic lung infection by P. aeruginosa. In the present study, the effects of ginseng on the formation of P. aeruginosa biofilms were further investigated in vitro and in vivo. Ginseng aqueous extract at concentrations of 0.5–2.0% did not inhibit the growth of P. aeruginosa, but significantly prevented P. aeruginosa from forming biofilm. Exposure to 0.5% ginseng aqueous extract for 24 h destroyed most 7-day-old mature biofilms formed by both mucoid and nonmucoid P. aeruginosa strains. Ginseng treatment enhanced swimming and twitching motility, but reduced swarming of P. aeruginosa at concentrations as low as 0.25%. Oral administration of ginseng extracts in mice promoted phagocytosis of P. aeruginosa PAO1 by airway phagocytes, but did not affect phagocytosis of a PAO1-filM mutant. Our study suggests that ginseng treatment may help to eradicate the biofilm-associated chronic infections caused by P. aeruginosa.

Introduction

Pseudomonas aeruginosa is a common bacterium frequently found in the environment. Pseudomonas aeruginosa causes opportunistic infections due to its virulence and its capacity to form biofilms (Lyczak et al., 2000). Chronic P. aeruginosa lung infection is the major cause of morbidity and mortality in cystic fibrosis (CF) patients (Høiby et al., 2005). This infection is highly resistant to antibiotic treatments and to host immune responses (Høiby et al., 2010). Intensive and aggressive antibiotic treatments may help to eradicate the intermittent P. aeruginosa lung colonization in CF patients, but it is impossible to eradicate the chronic infection once it has become established. The biofilm mode of growth is proposed to occur in the lungs of chronically infected CF patients and bacterial cells are thus protected from antibiotic treatment and the immune response (Høiby et al., 2001).

The mechanism of biofilm formation by P. aeruginosa has been investigated by many research groups. Extracellular polymeric substances, including polysaccharides, proteins and extracellular DNA, are important components that hold bacterial cells together, stabilize biofilm architecture and function as a matrix (Stoodley et al., 2002; Flemming et al., 2007). Type IV pili and flagella are required for P. aeruginosa biofilm formation (O'Toole & Kolter, 1998). Interactions between nonmotile and motile subpopulations of P. aeruginosa cells are involved in the formation of mushroom-shaped biofilm structures, which confer resistance to antibiotic treatments (Yang et al., 2007, 2009a, b; Pamp et al., 2008). Type IV pili are required for the motile subpopulation of P. aeruginosa cells to associate with extracellular DNA released from the nonmotile subpopulation of P. aeruginosa cells, and flagella-mediated chemotaxis is required for the movement of motile subpopulations of P. aeruginosa cells to nonmotile subpopulations of P. aeruginosa cells (Barken et al., 2008).

Thus, among the factors contributing to P. aeruginosa biofilm formation, type IV pili and flagella have proven to play essential roles. Pseudomonas aeruginosa can perform swimming motility in aqueous environments, which is mediated by its polar flagellum. In addition, two distinct types of surface-associated motility have been defined when P. aeruginosa grow on agar plates: twitching motility requiring functional type IV pili (Semmler et al., 1999; Mattick, 2002) and swarming motility requiring functional flagella, biosurfactant production and, under some conditions, type IV pili (Kohler et al., 2000; Deziel et al., 2003).

There is a strong interest in finding ways of inhibiting the development of biofilms or eliminating established biofilms. For example, iron chelators are used to prevent biofilm development, especially under low oxygen conditions such as in CF lungs with chronic infections of P. aeruginosa (O'May et al., 2009). Quorum-sensing inhibitors are used to block cell-to-cell communications and reduce biofilm formation by P. aeruginosa (Hentzer et al., 2003; Yang et al., 2009a, b). However, feasible methods to inhibit biofilm formation and eliminate established biofilms in vivo are limited. Therefore, it is important to develop novel therapies to combat biofilm-related infections, especially P. aeruginosa chronic lung infection.

Previously, we have demonstrated that Chinese ginseng could facilitate the clearance of an artificial biofilm infection in animal models and promote the development of a TH1 immune response that helps the infected hosts to clear the severe infection (Song et al., 1997a, b, 1998, 2003). In vitro studies suggested that ginseng exerts neither bactericidal nor inhibiting effects on P. aeruginosa (Song et al., 1997a, b, 2002). In the present study, we therefore investigated whether ginseng could affect P. aeruginosa biofilm in vitro, including both mucoid and nonmucoid strains, in flow chambers.

Materials and methods

Bacterial strains

The prototypic nonmucoid P. aeruginosa strain PAO1 (Holloway & Morgan, 1986) and its isogenic derivative stain mucoid PDO300 (Alg+ PAOmucA22) (Mathee et al., 1999), and mucoid P. aeruginosa NH57388A (Hoffmann et al., 2005), a clinical isolate from a CF patient, PAO1-filM (Klausen et al., 2003) and PAO1-pilA (Klausen et al., 2003) were used in the study.

Preparation of ginseng extract

The ginseng aqueous extract was prepared according to the method described previously (Song et al., 1997a, b). In brief, 2 g of Panax ginseng C.A. Meyer (Ginseng) powder (Ginseng age: 5–6 years, Jilin, China) was mixed with 100 mL of sterilized water. The mixture was heated for 30 min in a 90 °C water bath, and then centrifuged and sterile filtered using a disposable syringe filter (0.20 µm, Minisart Filters, Sartorius AG, 37070 Göttingen, Germany) before use. The final 2% ginseng extract contained total protein 2.47 mg mL−1, total polysaccharide 9.24 mg mL−1, and total Ginsenoside 1.98 mg mL−1. These parameters were used to adjust the quality of the ginseng extract.

Effects of ginseng on batch culture of P. aeruginosa

Pseudomonas aeruginosa wild-type PAO1 strain was cultivated in Luria–Bertani medium supplemented with different concentrations of ginseng extract at 37 °C under shaking conditions for 48 h. The culture results were generated by LabSystems Bioscreen C (FP-1100C, Finland) and the turbidity reflection of bacterial growth was expressed as OD.

Effects of ginseng on biofilm formation

To test the effects of ginseng treatment on biofilm formation and development, we grew P. aerguinosa biofilms in a medium supplemented with 0.5% ginseng. Gfp-tagged P. aerguinosa PAO1 and PDO300 (Hentzer et al., 2001) biofilms were grown at 30 °C in three-channel flow cells with individual channel dimensions of 0.3 × 4 × 40 mm (Sternberg & Tolker-Nielsen, 2006) supplied with AB minimal medium (Clark & Maaløe, 1967) with or without ginseng as a solvent described above and all media supplemented with 0.02% casamino acids (Lee et al., 2005). A microscope glass cover slip served as the substratum (Knittel 24 × 50 mm st1; Knittel Gläser, Braunschweig, Germany). Pseudomonas aeruginosa inocula from overnight culture diluted to an OD600 nm of 0.001 with 0.9% NaCl were used for inoculation of the flow channels. A Watson–Marlow 205S peristaltic pump was used to maintain the AB medium flow with or without 0.5% ginseng at a constant rate of 3 mL h−1.

Biofilm tolerance to tobramycin was assessed by supplementing the medium to the 3-day-old P. aeruginosa PAO1 and PDO300 biofilms with tobramycin at concentrations of 20 µg mL−1. The tobramycin treatments were continued for 24 h. Bacterial viability was assessed by staining ginseng-treated biofilms with 20 µM of propidium iodide for 10 min, followed by confocal laser scanning microscope (CLSM) observation.

Effects of ginseng on mature biofilms

The biofilms of P. aeruginosa PAO1, PDO300 and NH57388A were cultivated in flow chambers for 7 days. The tolerance of biofilms to ginseng was assessed by adding 0.5% ginseng to the influent medium of 7-day-old preformed biofilms for 24 h. Images were recorded from hour 0 to hour 24 under CLSM. Bacterial viability in biofilms was assessed using propidium iodide staining as described above.

Microscopy and image acquisition

All microscopic observations were performed on a Zeiss LSM510 confocal laser scanning microscope, CLSM (Carl Zeiss, Jena, Germany), equipped with an argon laser detector and filter sets for monitoring of green fluorescent protein (GFP) fluorescence. Images were obtained using a 40 ×/1.3 Plan-Neofluar Oil objective. Vertical cross-section images were generated using the imaris software package (Bitplane AG, Zurich, Switzerland).

Motility assays

  1. Swimming. Bacteria were inoculated using a sterile toothpick at the center of 5 mm ABT plates (AB medium containing 2.5 mg L−1 thiamine, 0.3% Bacto agar, 0.2% Casamino acids and 30 mM glucose). The swimming zone was measured after a 48-h incubation at room temperature.

  2. Twitching motility. Bacteria were stab inoculated with a toothpick through a thin 2-mm ABT medium supplemented with 0.2% Casamino acids, 30 mM glucose and 1.5% Bacto agar to the bottom of the Petri dish. After incubation for 24 and 48 h at 30 °C, the diameter of the hazy zone of growth was measured.

  3. Swarming. Bacteria were inoculated to swarm plates that were composed of 0.4% Bacto agar and ABT supplement with 0.5% Casamino acids and 0.5% glucose. The plates were dried for 2 h at room temperature. A total of 5 µL of an overnight culture was inoculated, the plates were incubated at 37 °C for 36 h and the surface locomotion of the bacteria was observed. All the motility assays were performed in triplicate.

Animal studies

Forty 12-week-old healthy female Balb/c mice were used in the study. The animals were divided into four groups and each contained 10 mice. Pseudomonas aeruginosa PAO1 and PAO1-filM were used as challenge strains, which were immobilized in alginate beads as described previously (Wu et al., 2001). The challenge concentrations were 108 CFU mL−1. Half the animals were fed with 5% ginseng aqueous extracts 2 h and 30 min before intratracheal challenge and the dosage were equal to 0.5% of the final concentration in animal body fluid. The other half of the animals functioned as a control and only received normal saline orally at the same timepoints. Each animal received 0.04 mL of PAO1 or PAO1-filM alginate beads intratracheally into the left lung on the basis of anesthesia using a mixture of fentanyl and fluanisone (Hypnorm, 10 mg mL−1) and Midazolam (Dormicum, 5 mg mL−1) at a ratio of 1 : 1. All animals were sacrificed at 24 h after challenge and bronchial alveolar lavage (BAL) was performed within 15 min. All BAL fluids were kept at 4 °C. The animal experiment was authorized by the National Animal Ethics Committee, Denmark.

Phagocytosis examination

BAL fluids were centrifuged to collect BAL cells. BAL smears were made and stained by Giemsa solution. Phagocytosis of BAL phagocytes was observed under a microscope to obtain the percentages of phagocytosis and the index of phagocytosis (average bacteria number phagocytized). The anova test was used to analyze the results of phagocytosis in the study.

Results

Ginseng extract does not affect the growth rate of P. aeruginosa

The growth of P. aeruginosa PAO1 was monitored for 48 h to determine any effect of ginseng on bacterial growth. Growth of the culture was monitored by OD measurements from inoculation to the stationary phase. The results showed that ginseng does not inhibit PAO1 growth, but if anything, had a weak stimulating effect (Fig. 1). Similar results were obtained with the mucoid strain of P. aeruginosa PDO300 and the clinical isolate of P. aeruginosa NH57388A (data not shown).

Figure 1

Growth of Pseudomonas aeruginosa PAO1 in Luria–Bertani media with different concentrations of ginseng at 37°C for 48 h. The curves show that ginseng did not inhibit bacterial growth.

Figure 1

Growth of Pseudomonas aeruginosa PAO1 in Luria–Bertani media with different concentrations of ginseng at 37°C for 48 h. The curves show that ginseng did not inhibit bacterial growth.

Ginseng extract reduces biofilm formation by P. aeruginosa

Nonmucoid P. aeruginosa wild-type PAO1 and its isogenic mucoid derivative PDO300 were cultured for 3 days in flow chambers in the presence or absence of 0.5% medium-supplemented ginseng extract. In the absence of ginseng, both mucoid and nonmucoid strains formed biofilms in the flow chambers, but the morphology of the biofilms of the two stains was different (Fig. 2). PAO1 formed a relatively flat biofilm, whereas PDO300 formed biofilms with distinct microcolonies. In contrast, the development of biofilms in both bacterial strains in the presence of 0.5% of ginseng was significantly inhibited (Fig. 2b and d). Moreover, biofilms formed by PAO1 and PDO300 without ginseng were tolerant to the treatment of tobramycin in 20 µg mL−1 for 24 h, whereas biofilms of the two strains developing poorly in the presence of 0.5% ginseng were sensitive to tobramycin, and most of the bacterial cells were eventually killed (Fig. 2b and d).

Figure 2

Green image shows the development of bacterial biofilms. Green biomass (GFP tagged) indicates live cells in flow chambers with or without 0.5% ginseng. On Day 3, tobramycin was added at a concentration of 20 µg mL−1 for 24 h, and the biofilm was stained on Day 4 by propidium iodide to show the dead cells in red color. Minimum inhibitory concentration of tobramycin for planktonic bacteria: 1 µg mL−1. (a) PAO1 control; (b) PAO1+ginseng; (c) PDO300 control; (d) PDO300+ginseng.

Figure 2

Green image shows the development of bacterial biofilms. Green biomass (GFP tagged) indicates live cells in flow chambers with or without 0.5% ginseng. On Day 3, tobramycin was added at a concentration of 20 µg mL−1 for 24 h, and the biofilm was stained on Day 4 by propidium iodide to show the dead cells in red color. Minimum inhibitory concentration of tobramycin for planktonic bacteria: 1 µg mL−1. (a) PAO1 control; (b) PAO1+ginseng; (c) PDO300 control; (d) PDO300+ginseng.

Ginseng extract dispersed preformed biofilms of P. aeruginosa

Biofilms of wild-type PAO1, mucoid PDO300 and a mucoid clinical isolate NH57388A were developed in flow chambers for 7 days, after which the medium was supplemented with 0.5% ginseng extract. Surprisingly, after exposure to the ginseng-supplemented medium, the biofilms of the three stains were gradually removed with few or no live bacteria after 20 h of exposure to ginseng (Fig. 3). The biofilm of nonmucoid wild-type PAO1 showed nearly no living bacterial cells after 10 h of exposure to the ginseng extract (Fig. 3a). The PAO1 biofilm disappeared much faster than the two mucoid biofilms (Fig. 3b and c). Constant observations under CLSM revealed that a rapid movement and dissolution of the cellular mass took place inside the preformed biofilms. This phenomenon was observed for all strains including the clinical isolate of NH57388A. The motility of the P. aeruginosa bacterial cells was in general elevated after exposure to ginseng (data not shown).

Figure 3

Preformed 7-day-old biofilm interacted with 0.5% of ginseng for 10–20 h meanwhile stained by propidium iodide to show the dead cells in red color. (a) PAO1 biofilm was treated for 10 h with ginseng. (b) PDO300 biofilm was exposed for 20 h to ginseng. (c) NH53788A preformed biofilm, a clinic mucoid isolate, was exposed for 20 h to ginseng.

Figure 3

Preformed 7-day-old biofilm interacted with 0.5% of ginseng for 10–20 h meanwhile stained by propidium iodide to show the dead cells in red color. (a) PAO1 biofilm was treated for 10 h with ginseng. (b) PDO300 biofilm was exposed for 20 h to ginseng. (c) NH53788A preformed biofilm, a clinic mucoid isolate, was exposed for 20 h to ginseng.

Ginseng extract enhances swimming and twitching motility, but reduces swarming motility

Swarming motility has been characterized as flagella-dependent movement on viscous surfaces. The effect of 0.25% of ginseng on the swarming motility of P. aeruginosa PAO1, the isogenic fliM mutant and the mucoid PDO300 was evaluated. Swarming was only observed in the plate of PAO1 in the absence of ginseng. This result suggests that ginseng reduces the swarming motility of P. aeruginosa PAO1 (Fig. 4a).

Figure 4

Effects of ginseng on Pseudomonas aeruginosa motilities in vitro. (a) Inhibition of swarming motility in P. aeruginosa by ginseng. Significant swarming motility is seen in the PAO1 plate (the middle of upper panel). Ginseng (0.25%) is sufficient to inhibit the swarming motility of PAO1 (the middle lower panel). (b) Stimulation of swimming motility in P. aeruginosa by ginseng. The swimming motility of both PAO1 and PDO300 was significantly stimulated by 0.25% of ginseng (the lower panel). (c) Activation of twitching motility in P. aeruginosa by ginseng. The twitching motility (indicated by red arrows) of both PAO1 and PDO300 was significantly activated by 0.25% of ginseng.

Figure 4

Effects of ginseng on Pseudomonas aeruginosa motilities in vitro. (a) Inhibition of swarming motility in P. aeruginosa by ginseng. Significant swarming motility is seen in the PAO1 plate (the middle of upper panel). Ginseng (0.25%) is sufficient to inhibit the swarming motility of PAO1 (the middle lower panel). (b) Stimulation of swimming motility in P. aeruginosa by ginseng. The swimming motility of both PAO1 and PDO300 was significantly stimulated by 0.25% of ginseng (the lower panel). (c) Activation of twitching motility in P. aeruginosa by ginseng. The twitching motility (indicated by red arrows) of both PAO1 and PDO300 was significantly activated by 0.25% of ginseng.

The swimming motility of P. aeruginosa also depends on flagellar movement. Activated swimming was observed for PAO1 in the presence of ginseng. Furthermore, ginseng could clearly also facilitate swimming of the mucoid PDO300. As expected, the fliM mutant did not show any swimming motility in either condition (Fig. 4b).

Twitching motility is caused by type IV pili-mediated bacterial translocation on a solid surface. Therefore, a pilA mutant was used as a negative control (Fig. 4c). Ginseng clearly induced twitching motility of both PAO1 and PDO300. The twitching motility of PAO1 was activated more than that of PDO300.

Ginseng extract promotes phagocytosis of P. aeruginosa by mouse airway phagocytes

The phagocytosis rate and index are expressed as Median (range) in the study. Twenty-four hours after intratracheal challenge, no significant differences were seen in both the phagocytosis rate and index between the PAO1-filM control and ginseng-treated groups (P>0.27 and >0.8). However, in the PAO1-infected animals, ginseng-treated BAL phagocytes showed a significantly higher phagocytosis rate (P=0.0004) and index (P<0.01) compared with the control animals (Fig. 5a and b).

Figure 5

Effects of ginseng on Pseudomonas aeruginosa motilities in vivo. (a) In the flagella-deficient P. aeruginosa PAO1-filM-infected animals, no significant difference in phagocytosis was seen between the animals treated with and without ginseng. However, in the PAO1 wild-type infected animals, the ginseng-treated group showed significantly higher phagocytosis rates than the control animals. (b) Twenty-four hours after intratracheal challenge, significantly improved phagocytosis index was found in the ginseng-treated P. aeruginosa PAO1-infected animals compared with the control animals. However, in the flagella-deficient PAO1-filM-infected animals, ginseng treatment did not improve the phagocytosis index.

Figure 5

Effects of ginseng on Pseudomonas aeruginosa motilities in vivo. (a) In the flagella-deficient P. aeruginosa PAO1-filM-infected animals, no significant difference in phagocytosis was seen between the animals treated with and without ginseng. However, in the PAO1 wild-type infected animals, the ginseng-treated group showed significantly higher phagocytosis rates than the control animals. (b) Twenty-four hours after intratracheal challenge, significantly improved phagocytosis index was found in the ginseng-treated P. aeruginosa PAO1-infected animals compared with the control animals. However, in the flagella-deficient PAO1-filM-infected animals, ginseng treatment did not improve the phagocytosis index.

Discussion

The biofilm mode of growth of P. aeruginosa in CF airways is associated with significant tolerance to antibiotics and the immune responses (Stewart & Costerton, 2001; Høiby et al., 2010). Biofilm formation of P. aeruginosa requires both type IV pili and flagella-mediated motility (O'Toole & Kolter, 1998). More recently, type IV pili (but not the pili-associated motility) were shown to be required for interactions with extracellular DNA during the development of mature P. aeruginosa biofilm structures (Barken et al., 2008). In fact, excess twitching motility leads to a reduction of biofilm formation by P. aeruginosa (Singh et al., 2002). In contrast to twitching motility, flagella-mediated motility is required for the development of mature P. aeruginosa biofilm structures (Barken et al., 2008).

The present study shows that ginseng does not inhibit the growth of P. aeruginosa (Fig. 1), but it prevents the efficient development of P. aeruginosa biofilms in vitro (Fig. 2). Furthermore, preformed 7-day-old biofilms, including mucoid and nonmucoid laboratory strains and a clinical isolate, are almost completely dispersed within 24 h after exposure to ginseng extracts (Fig. 3). We have observed extensive cell movement in the microcolonies of biofilms treated with ginseng extracts (data not shown), which may result in cells migrating out of the preformed biofilms in accordance with the results from the swimming and twitching tests (Fig. 4b and c). These results indicate that flagellum-mediated swimming motility is not required for P. aeruginosa biofilm structure development. The presence of several dead bacterial cells in the biofilms after exposure to ginseng extract suggests that ginseng extract also activates apoptosis-like mechanisms in the biofilm cells (Fig. 3). We have also demonstrated in another study that such effects of ginseng are not dominated by ginseng saponins (data not shown).

Swarming motility is essential for biofilm development (Shrout et al., 2006; Pamp & Tolker-Nielsen, 2007). Moreover, swarming motility has been shown to be part of a complex differentiation process, which leads to increased production of virulence factors and antibiotic resistance (Overhage et al., 2008). Swarming is dependent on functional quorum sensing (which induces the production of rhamnolipid), type IV pili and flagella (Kohler et al., 2000; Deziel et al., 2003). We have demonstrated recently that ginseng extract reduces the production of signal molecules of quorum sensing (BHL and OdDHL) in supernatants of P. aeruginosa PAO1 cultures (Song et al., 2010). This finding may partly explain our results from the swarming tests in this study. However, the molecular mechanism of inhibition of swarming motility and induction of swimming and twitching motility by ginseng extract is still unknown and needs further studies.

In our animal study, pretreatment with ginseng orally resulted in significantly higher phagocytosis rates and index in the BAL phagocytes from the wild-type P. aeruginosa PAO1-infected animals compared with saline-pretreated animals (Fig. 5a and b). In contrast, in the animals infected with flagella-deficient P. aeruginosa PAO1-filM, ginseng pretreatment did not improve the phagocytosis or the index. Clearly, the significantly increased phagocytosis rate and index in the PAO1-infected animals are due to the stimulation of P. aeruginosa PAO1 motility induced by ginseng in vivo.

Previously, we demonstrated in our animal models of chronic P. aeruginosa lung infection that ginseng treatment results in faster bacterial clearance from the lungs and milder lung pathology when compared with the untreated animals (Song et al., 1997a, b, 1998). We also observed a significantly stronger neutrophil chemiluminescence in the blood, a shift of the immune response from a high anti-P. aeruginosa immunoglobulin G (IgG) response and local infiltration of mast cells in the lungs (T-helper type 2 response) to a TH1 immune response characterized by downregulation of IgG and upregulation of IgG2a levels, and improved functions of phagocytes by means of upregulated production of interferon-γ and downregulated interleukin-4 in the lung tissues and spleen (Song et al., 1997a, b, 1998, 2003, 2005). It has been well documented that a TH1 response favors host cleaning of infections by P. aeruginosa (Johansen et al., 1995, 1996., 1997; Moser et al., 1997, 2000, 2005).

Our results from the present study suggest that ginseng induces increased bacterial motility in the biofilm-like alginate beads, resulting in the release of bacteria from the biofilm and loss of protective effects from the polymeric matrix, followed by an increased efficiency of the host immune system and antibiotics to clear the biofilm infection. The activation of the TH1 immune response induced by ginseng treatment and the increased motility of bacteria due to the effects of ginseng might exhibit a synergistic effect on the infection. However, the activation of P. aeruginosa motility might theoretically result in spreading of the infection in the body, which needs to be clarified in our next study.

Ginseng is one of the traditional Chinese medicines that is widely used not only in China, Korea and Japan but also in the rest of the world, including Europe and North America. It is well accepted in China that ginseng is a tonic medicine with multiple modulating effects on different organ systems in the human body (Huang, 1993). Our previous animal studies showed that ginseng has therapeutic effects on chronic P. aeruginosa lung infections by inducing a TH1-dominated immune response. The present results suggest that ginseng can disturb the development of P. aeruginosa biofilm and cause dissolution of mature biofilms, most likely by activating the motility of P. aeruginosa. Apparently, ginseng is a potentially promising remedy for the treatment of P. aeruginosa biofilm infections.

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Author notes

Editor: Richard Marconi