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Keywords:

  • cyclodextrins;
  • enzyme mimics;
  • glutathione peroxidase;
  • selenium;
  • substrate binding

Abstract

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusion
  5. Experimental procedures
  6. Acknowledgements
  7. References

A 6A,6A′-dicyclohexylamine-6B,6B′-diselenide-bis-β-cyclodextrin (6-CySeCD) was designed and synthesized to imitate the antioxidant enzyme glutathione peroxidase (GPX). In this novel GPX model, β-cyclodextrin provided a hydrophobic environment for substrate binding within its cavity, and a cyclohexylamine group was incorporated into cyclodextrin in proximity to the catalytic selenium in order to increase the stability of the nucleophilic intermediate selenolate. 6-CySeCD exhibits better GPX activity than 6,6′-diselenide-bis-cyclodextrin (6-SeCD) and 2-phenyl-1,2-benzoisoselenazol-3(2H)-one (Ebselen) in the reduction of H2O2, tert-butyl hydroperoxide and cumenyl hydroperoxide by glutathione, respectively. A ping-pong mechanism was observed in steady-state kinetic studies on 6-CySeCD-catalyzed reactions. The enzymatic properties showed that there are two major factors for improving the catalytic efficiency of GPX mimics. First, the substrate-binding site should match the size and shape of the substrate and second, incorporation of an imido-group increases the stability of selenolate in the catalytic cycle. More efficient antioxidant ability compared with 6-SeCD and Ebselen was also seen in the ferrous sulfate/ascorbate-induced mitochondria damage system, and this implies its prospective therapeutic application.

Abbreviations
BHT

2,6-di-tert-butyl-4-methylphenol

β-CD

β-cyclodextrin

6-CySeCD

6A,6A′-dicyclohexylamine-6B,6B′-diselenide-bis-β-cyclodextrin

CumOOH

cumenyl hydroperoxide

Ebselen

2-phenyl-1,2-benzoisoselenazol-3(2H)-one

GPX

glutathione peroxidase

GSH

glutathione

6-SeCD

6,6′-diselenide-bis-cyclodextrin

TBARS

thiobarbituric acid reactive substances

t-BuOOH

tert-butyl hydroperoxide

Glutathione peroxidase (GPX; EC 1.11.1.9) is a well-known selenoenzyme that catalyzes the reduction of harmful hydroperoxides by glutathione (GSH) (Scheme 1) and protects lipid membranes and other cellular components against oxidative damage [1–4]. It is related to many diseases and is regarded as one of the most important antioxidant enzymes in living organisms. GPX enzyme activity is sometimes increased in disease, possibly as a compensatory mechanism to try to counteract the oxidative stress associated with the pathology, although it is also decreased in other diseases [5–8]. Therefore, modulation of GPX may be involved in many pathological conditions. Because natural GPX has some shortcomings, such as instability, antigenicity and poor availability, much attention has been paid to its artificial imitation [9,10].

image

Figure Scheme 1. . Catalytic cycle for GPX.

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In synthetic approaches, an initial attempt is made to synthesize organoselenium compounds in which the interaction of Se–N in GPX catalysis is imitated by inducing N or O in close proximity to selenium. One way in which this is achieved is by binding the selenium atom directly to a heteroatom such as nitrogen. 2-Phenyl-l,2-benzisoselenazol-3(2H)-one (Ebselen), the first biologically active organoselenium compound, represents an excellent example of a GPX mimic [9]. Another way to imitate the Se···N interaction in GPX is if the selenium is not bound directly to the heteroatom (N or O), but is located in close proximity to it, this approach seems to help stabilize the selenolate and enhance the GPX-like activity of diselenides [11]. Although some GPX mimics show some increased activity, most show only limited catalytic enhancement. Based on an understanding of the structure of GPX, its mode of molecular recognition and catalysis, as well as previous studies [12–14], we believe that generation of specific binding ability for the thiol substrate and correct incorporation of the functional selenium group should be critical in the construction of an effective GPX model. Previous studies by our group in preparing GPX models using a mAb technique [15,16], bioimprinting [17] and the chemical modification of native enzymes [18] have supported this hypothesis. Recently, we developed some GPX mimics in which the β-cyclodextrin (β-CD) cavity provided a hydrophobic environment for substrate binding [19–23]. For example, 6,6’-diseleno-bis-cyclodextrin (6-SeCD) activity for the reduction of hydrogen peroxide (H2O2) by GSH is 4.3 times that of Ebselen because of the role of the hydrophobic cavity of β-CD in binding substrate.

In this study we designed and synthesized a new GPX mimic, 6A,6A′-dicyclohexylamine-6B,6B′-diselenide-bis-β-cyclodextrin (6-CySeCD), in which the cyclohexylamine group was incorporated in the proximity of the selenium atom and the β-CD cavity provided a hydrophobic environment for substrate binding. 6-CySeCD showed higher GPX activity than 6-SeCD for the reduction of H2O2, tert-butyl hydroperoxide (t-BuOOH) and cumenyl hydroperoxide (CumOOH) by GSH, indicating that incorporation of the imido-group in the proximity of the selenium atom may increase the stability of the nucleophilic intermediate selenolate and enhance GPX-like activity in selenium-containing GPX mimics. We also studied the catalytic mechanism using steady-state kinetics of 6-CySeCD catalysis and investigated the antioxidant ability of 6-CySeCD using a mitochondria injury system.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Synthesis and characterization of 6-CySeCD

The synthetic routes of 6-CySeCD are shown in Scheme 2. 6-CySeCD was analyzed using elemental analysis, found (calculated for C96H160O66N2Se2·6H2O) %: C, 43.67 (43.28); H, 6.49 (6.31); N, 1.06 (1.05). IR (KBr): 3376(-OH), 2926(CH,CH2), 1644(-OH), 1568, 1467(-NH-), 1158, 1079, 1031(-O-), 948, 840, 755, 709, 579 cm−1. 13C NMR (400 MHz, D2O) δ(p.p.m.): 102.5(C1), 81.9(C4), 73.3(C3), 72.7(C5), 72.0(C2), 60.3(C6), 59.2(C6′), 52.3(C7), 35.1(C8), 28.5(C10), 25.7(C9).

image

Figure Scheme 2. . Synthetic route of 6-CySeCD.

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The content and valence of selenium in 6-CySeCD were measured by X-ray photoelectron spectroscopy. The Se3d electronic-binding energy of 6-CySeCD is 54.9 eV, which approaches the binding energy of SeCys (55.1 eV), indicating that the selenium in 6-CySeCD is present in the form of −1 valence (diselenium bridge, -Se-Se-). The experiment also gave the C/Se ratio, which is 48.3 : 1 (calculated 48 : 1), indicating 2 mol of selenium per mol of mimic. Thus, the structure of 6-CySeCD should be as shown in Scheme 3.

image

Figure Scheme 3. . Structures of 6-CySeCD.

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GPX activity of 6-CySeCD

The initial reaction rate for the reduction of hydroperoxides by GSH was determined by observing the change in NADPH absorption at 340 nm (Eqns 1,2). The GPX activities of 6-CySeCD and other GPX mimics catalyzed the reduction of hydroperoxides by GSH are listed in Table 1.

  • image(1)
  • image(2)

The GPX activities of 6-CySeCD and 6-SeCD for the reduction of H2O2 by GSH were 7.9 and 4.2 min−1, respectively, indicating that 6-CySeCD and 6-SeCD display higher GPX activity than Ebselen. This result is not surprising, because β-CD shows good substrate binding compared with Ebselen [15]. When the substrates were H2O2, t-BuOOH and CumOOH, we found that the GPX activities with 6-CySeCD for the reduction of H2O2, t-BuOOH and CumOOH by GSH were higher than with 6-SeCD.

Table 1.   Comparison between GPX activities of the 6-CySeCD-catalyzed reduction of hydroperoxides by GSH and other species. One unit of enzyme activity is defined as amount of mimic that utilizes of 1 μmol of NADPH per minute. All data are presented as means ± SD.
MimicsHydroperoxideActivity (min−1)
  1. a  Reactions were carried out in 50 mm potassium phosphate buffer, pH 7.0, at 37 °C, 1 mm GSH, 0.5 mm hydroperoxide. b Obtained from Liu et al. [19].

EbselenH2O20.99b
6-SeCDH2O24.20 ± 0.15b
t-BuOOH6.3 ± 0.2
CumOOH10.7 ± 0.4
6-CySeCDaH2O27.9 ± 0.4
t-BuOOH12.3 ± 0.3
CumOOH18.3 ± 0.5

In the investigation of GPX mimics, Wilson's diselenides are successful [11]. As shown in Scheme 4, there are two processes (oxidation and reduction with thiols) in the mechanism, and the N near the selenium moiety apparently helps stabilize the selenolate and enhance the GPX-like activity of diselenides. In this study, a cyclohexylamine group was incorporated in the proximity of the active selenium atom in the 6-CySeCD molecule and the GPX activity for 6-CySeCD was higher than for 6-SeCD.

image

Figure Scheme 4. . Catalytic mechanism proposed by Wilson et al.[11].

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Kinetics of 6-CySeCD

Steady-state kinetics was observed for substrates H2O2 and GSH. The initial velocities for reduction of H2O2 by GSH were determined as a function of the substrate concentration at 37 °C and pH 7.0, by varying one substrate concentration while another was fixed. The relevant steady-state equation (Eqn 3) for the mimic reaction is

  • image(3)

Where v0 is the initial reaction rate, [E]0 is the initial enzyme mimic concentration, kmax is a pseudo-first-order rate constant KH2O2 and KGSH are the Michaelis–Menten constants (Km) for H2O2 and GSH, respectively. Double reciprocal plots of the initial velocity versus the concentration of substrates gave a family of parallel lines (Fig. 1), indicating that the reaction mechanism is a ping-pong mechanism. This result demonstrated that the GPX mimic, 6-CySeCD, has the same catalytic mechanism as native GPX. From the steady-state equation, the kinetic parameters were obtained (Table 2).

image

Figure 1.  Double-reciprocal plots for the reduction of H2O2 by GSH catalyzed by 5 μm 6-CySeCD. (A) [E]0/v0 versus 1/[H2O2] (mm−1) at [GSH] 0.5 (▪), 1 (•) and 3 mm (bsl00072). (B) [E]0/v0 versus 1/[GSH] (mm−1) at [H2O2] 0.5 (•), 1 (bsl00066) and 2 mm (bsl00072).

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Table 2.   Kinetic parameters of the 6-CySeCD. Reactions were carried out in 50 mm potassium phosphate buffer, pH 7.0, at 37 °C, 0.5–3.0 mm GSH, 0.5–2.0 mm H2O2. All data are presented as means ± SD.
GPX mimickmax (min-1)KGSH (mm)kmax/KGSH (m−1·min-1)KH2O2m)kmax/KH2O2 (m−1·min−1)
6-CySeCD18.3 ± 0.51.63 ± 0.14(1.12 ± 0.22) × 103547 ± 13(3.35 ± 0.17) × 104

During investigation of the reduction of peroxides, it is natural to consider the possibility of free radical reactions. Bell and Hilvert [24] used a radical trap, 2,6-di-tert-butyl-1-4-methylphenol (BHT), to inhibit the reduction of t-BuOOH by a thioyl compound in the presence of a GPX mimic, selenosubtilisin, and showed that the enzyme-catalyzed reduction of hydroperoxide proceeds via a nonradical mechanism, although the spontaneous reduction of hydroperoxide by GSH involves the production of free radicals. The same results were found for the 6-CySeCD-catalyzed reduction of H2O2 by GSH. BHT inhibited the spontaneous reaction, but not the 6-CySeCD-catalyzed reduction (Fig. 2). This suggested that 6-CySeCD also catalyzes the reduction of hydroperoxide by GSH via a nonradical mechanism.

image

Figure 2.  Plots of v0 versus [H2O2] for 1 mm GSH in 50 mm potassium phosphate buffer, pH 7.4, and 37 °C, at [BHT] = 0 μm (a) and 50 μm (b). (A) [6-CySeCD], 0 μm; (B) [6-CySeCD], 5 μm.

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Protection of mitochondria against oxidative damage by 6-CySeCD

The swelling and shrinking of mitochondria are normal physiological phenomena during respiration. However, abnormal swelling disrupts the mitochondrial membrane resulting in cell death. Mitochondrial swelling therefore characterizes its integrity. Figure 3A shows that the mitochondrial swelling is greatly increased by ferrous sulfate/ascorbate-induced mitochondrial damage and the swelling is decreased by addition of 6-CySeCD.

image

Figure 3.   (A) Effect of concentration of 6-CySeCD on the swelling of mitochondria. (a) Control; (b) damage + 20 μm 6-CySeCD; (c) damage + 10 μm 6-CySeCD; (d) damage + 4 μm of 6-CySeCD; (e) damage. (B) Effect of different GPX mimics on mitochondrial swelling. (a) Control; (b) damage + 10 μm 6-CySeCD; (c) damage + 10 μm 6-SeCD; (d) damage + 10 μm of Ebselen; (e) damage.

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The absorbance at 520 nm for the control group was basically constant, whereas that for the damage group decreasede considerably over time, indicating that mitochondrial swelling was increased. However, the swelling in the protection group, which contained a certain concentration of 6-CySeCD, was apparently decreased compared with the damage group, and the mitochondrial swelling decreased with increasing 6-CySeCD concentration. The ability of GPX mimics (6-CySeCD, 6-SeCD and Ebselen) to protect mitochondria differed, as shown in Fig. 3B, and 6-CySeCD was the best among them. This is in agreement with ability of these GPX mimics to remove H2O2.

In this study, we used thiobarbituric acid reactive substances (TBARS) as a marker for lipid peroxidation, and 6-CySeCD, 6-SeCD and Ebselen as antioxidants in ferrous sulfate/ascorbate-induced mitochondrial damage to determine the levels of lipid peroxidation. Figure 4A shows the extent of protection afforded by 6-CySeCD. The amount of TBARS seen during mitochondrial damage was reduced considerably in the presence of 6-CySeCD, and the amount of TBARS decreased with increasing 6-CySeCD concentration. When the 6-CySeCD concentration was 20 µm and mitochondria were damaged for 50 min, the TBARS content was only 24% of that in the damage group without 6-CySeCD, indicating that 76% of TBARS production was inhibited. In order to gauge the ability of the three GPX mimics (6-CySeCD, 6-SeCD, and Ebselen) to inhibit TBARS production, their antioxidant activities were compared under identical conditions. As shown in Fig. 4B, the ability of 6-CySeCD to decrease the accumulation of TBARS was greater than that of 6-SeCD and Ebselen. In addition, we also tested the effect of 20 µm 6-CySeCD in the absence of damage (data no shown) and the result shows that 20 µm 6-CySeCD did not have any effect on mitochondrial swelling and lipid peroxidaton in the absence of damage.

image

Figure 4.   (A) Dependence of extent of TBARS accumulation on the concentration of 6-CySeCD. (a) Control; (b) damage + 20 μm 6-CySeCD; (c) damage + 10 μm 6-CySeCD; (d) damage + 4 μm 6-CySeCD; (e) damage. (B) Effect of different GPX mimics on TBARS accumulated during mitochondrial damage. (a) Control; (b) damage + 10 μm 6-CySeCD; (c) damage + 10 μm 6-SeCD; (d) damage + 10 μm Ebselen; (e) damage. Relative TBARS content calculated based on amount of TBARS for 50 min with damage group = 1.

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Exposing mitochondria in vitro to redox active xenobiotics may simulate oxidative damage of mitochondria in vivo. The reactions for ferrous sulfate/ascorbate-inducing mitochondrial damage can be proposed as follows:

  • image(4)
  • image(5)
  • image(6)

where H2O2 was produced by oxidation of ascorbic acid to dehydroascorbic acid (Eqn 4)[22], in addition, mitochondria can produce superoxide by Fe(II), which could be dismutated by mitochondrial superoxide dismutase to hydrogen peroxide. A hydroxyl radical was produced via the Fenton reaction (Eqns 5,6)[25–27]. The biological molecules in mitochondria are easily attacked by hydroxyl radicals, when changes in composition, morphology, structure, integrity, and function of the mitochondria take place. GPX mimics can scavenge hydroperoxides and block hydroxyl radical production, therefore protecting mitochondria against oxidative damage.

In the ferrous sulfate/ascorbate-induced mitochondrial damage model system, swelling and TBARS content were chosen according to the standard, which was used to determine the injury and extent of protection in mitochondria. 6-CySeCD reduced the mitochondrial swelling during damage and decreased the maximal TBARS content. Mitochondrial swelling and the amount of TBARS were decreased in a dose-dependent manner by 6-CySeCD. The inhibited TBARS content and decreased mitochondrial swelling can be explained by 6-CySeCD acting as a GPX mimic, which effectively scavenged hydroperoxide and protected mitochondria against oxidative damage.

Conclusion

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusion
  5. Experimental procedures
  6. Acknowledgements
  7. References

We developed a novel GPX mimic, 6-CySeCD. In this enzyme model, the cavity of β-CD supplied a hydrophobic environment for substrate binding, and mimic activity was increased greatly by the incorporation of a cyclohexylamine group in the proximity of the active selenium atom. Compared with Ebselen and 6-SeCD, 6-CySeCD is a better GPX mimic, as evidenced by its enzymatic properties and protection of mitochondria. These studies show that there are two key factors for improving catalytic efficiency of GPX mimics. First, the substrate-binding site should match the size and shape of the substrates, and second, incorporation of an imido-group increases the stability of transition state selenolate in the catalytic cycle. We believe that this is significant when designing mimics with high catalytic efficiency.

Experimental procedures

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusion
  5. Experimental procedures
  6. Acknowledgements
  7. References

Apparatus and materials

Structural characterization of 6-CySeCD was performed with a IFS-FT66V infrared spectrometer (Bruker, Bremen, Germany), a Varian Unity-400 NMR spectrometer (Varian Inc, Palo Alto, CA) and a Perkin-Elmer 240 DS elemental analyzer (Wellesley, MA). The content and valence of selenium in the 6-CySeCD were determined by using an ESCALAB MKII X-ray photoelectron spectrometer (VG Scientific, Sussex, UK). Spectrometric measurements were carried out by using a Shimadzu UV-2550 spectrophotometer (Kyoto, Japan).

β-CD was purchased from Shanghai Sanpu Chemical Plant (Shanghai, China), recrystallized three times from water and dried for 12 h at 120 °C in vacuum. Sodium borohydride, selenium, BHT and 1,3-benzene-disulfonyl chloride were obtained from Sigma (St Louis, MO). GSH, glutathione reductase, t-BuOOH, CumOOH, and NADPH were also obtained from Sigma. Sephadex G-25 was purchased from Pharmacia (Uppsala, Sweden). All the other materials were of analytical grade and obtained from Beijing Chemical Plant (Beijing, China).

Synthesis of 6-CySeCD

Two grams of 6A,6B-diiodo-6A,6B-dideoxy-β-cyclodextrin [28] was dissolved in 30 mL of dry dimethylformamide, and 185 µL of cyclohexylamine was added. The mixture was stirred at 45 °C for 4 h, dimethylformamide was evaporated under reduced pressure. The residue was dissolved in 45 mL potassium phosphate buffer (50 mm, pH 7.0) and 30 mL dimethylformamide (cosolvent). Then 7 mL of 1 m sodium hydroselenide (NaHSe), prepared according to the procedure of Klayman and Griffin [29] was added under the pure nitrogen. The mixture was kept under nitrogen for 36 h at 60 °C, oxidized in air and finally purified by centrifugation and Sephadex G-25 column (Φ2 × A60 cm) chromatography (λ = 254 nm) with distilled water as the eluent. The resulting solution was freeze-dried and the lyophilized powder was washed with ethyl ether three times and dried under vacuum to obtain a light yellow, pure sample with 34% yield. The structure of 6-CySeCD was analyzed by means of elemental analysis, IR, 13C NMR. The content and valence of selenium in the 6-CySeCD was determined by X-ray photoelectron spectroscopy. The energy of the exciting X-ray was 1253.6 eV (Mg, Kα). C1s = 285.0 eV served as standard. Scans were performed 12 times.

Determination of GPX-like activity and kinetics

Catalytic activities were determined by the method of Wilson et al. [11]. The reaction was carried out at 37 °C in 700 µL of solution containing 50 mm, pH 7.0, potassium phosphate buffer, 1 mm EDTA, 1 mm sodium azide, 1 mm GSH, 0.25 mm NADPH, 1 unit glutathione reductase, 5 µm 6-SeCD and 6-CySeCD. The reaction was initiated by addition of 0.5 mm hydroperoxide. Organic hydroperoxides (t-BuOOH, CumOOH) were dissolved in 0.2% (v/v) Triton X-100, which was the cosolvent and did not affect the GPX-like activity assay. Activity was determined by the decrease of NADPH absorption at 340 nm (εNADPH = 6220 m−1 cm−1). Background absorption of the noncatalytic reaction was run without mimic and was subtracted. The activity unit of enzyme mimic was defined as the amount of enzyme mimic, which utilizes 1 µmol NADPH per min.

The assay of 6-CySeCD kinetics was similar to that for native GPX [30]. Initial reduction rates of H2O2 by GSH were determined by observing the change in NADPH absorption at 340 nm at 37 °C and pH 7.0, varying one substrate concentration while another is fixed. All kinetic experiments were performed at 37 °C in 700 µL of reaction solution containing 0.5–3.0 mm GSH, 0.5–2.0 mm H2O2, 50 mm potassium phosphate buffer (pH 7.0), 1 mm EDTA, 0.25 mm NAPDH, 1 unit GSH reductase and 5 µm 6-CySeCD. Background absorption of the noncatalytic reaction was run without mimic and was subtracted. Kinetic data were analyzed by double-reciprocal plotting.

Preparation of mitochondria

Bovine heart mitochondria were isolated from fresh bovine heart [31] and suspended in 0.25 m sucrose, 10 mm EDTA and 25 mm Hepes-NaOH buffer, pH 7.4, and maintained on ice. The concentration of the mitochondrial protein was determined by Coomassie Brilliant Blue [32] using BSA as the standard.

Ferrous sulfate/ascorbate-induced mitochondrial damage

Mitochondria (0.5 mg protein·mL−1) suspended in medium (0.125 m KCl, 1 mm MgCl2, 5 mm glutamate, 1 mm GSH, 10 mm potassium phosphate buffer, pH 7.4) were subjected, in the absence and presence of the mimic, to oxidative stress generated by 0.5 mm ascorbate plus 12.5 µm ferrous sulfate at 37 °C. Damage experiments were carried out without mimic and known as the damage group; the experiment carried out without the mimic, ascorbate, and ferrous sulfate was known as the control group.

Biological analysis of mimics against mitochondrial damage

Mitochondrial swelling was assayed as described by Hunter et al. [33]. Mitochondrial swelling was measured by the decrease in the turbidity of the reaction mixture at 520 nm. The decrease in absorbance indicated an increase in mitochondrial swelling and a decrease in mitochondria integrity.

TBARS content in ferrous sulfate/ascorbate-treated mitochondria was analyzed by thiobarbituric acid assay [34]. In this assay, thiobarbituric acid reacts with malonaldehyde and/or other carbonyl by-products of free-radical-mediated lipid peroxidation to give 2 : 1 (mol/mol) colored conjugates, which have an A532 value.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusion
  5. Experimental procedures
  6. Acknowledgements
  7. References

This research was supported by Natural Science Foundation of China (Project no. 20572035 and 20534030) and Jilin University, Changchun, China.

References

  1. Top of page
  2. Abstract
  3. Results and Discussion
  4. Conclusion
  5. Experimental procedures
  6. Acknowledgements
  7. References
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