|تعداد مشاهده مقاله||111,487,493|
|تعداد دریافت فایل اصل مقاله||86,125,662|
A New Practical Purification Method for Type D Clostridium perfringens Epsilon Toxin by Size-Exclusion Chromatography (SEC) and Ultrafiltration (UF)
|Iranian Journal of Veterinary Medicine|
|مقاله 9، دوره 16، شماره 2، تیر 2022، صفحه 178-187 اصل مقاله (509.64 K)|
|نوع مقاله: Genetic|
|شناسه دیجیتال (DOI): 10.22059/ijvm.2022.83107|
|Mokarameh Poudineh Morref* 1؛ Mohammad Kazem Koohi1؛ Mojtaba Alimolaei2؛ Tara Emami3؛ Jalal Hassan1|
|1Department of Comparative Biosciences, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran|
|2Department of Research and Technology, Kerman branch, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Kerman, Iran|
|3Department of Proteomics and Biochemistry, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Karaj, Iran|
BACKGROUND: The high potential toxicity of epsilon toxin (Etx) produced by Clostridium perfringens (C. perfringens) type D, has made it the third most lethal clostridial toxin behind botulinum and tetanus, therefore, having a pure and concentrated Etx is very important.
OBJECTIVES: The aim of this study was to purify Etx as pure as possible with an applicable, cost-effective, multistep purification protocol with the lowest and shortest time.
METHODS: The purification of the Etx was carried out in multiple consecutive steps; ammonium sulfate precipi-tation, dialysis, size exclusion chromatography by G50, two concentration steps, and ultrafiltration. The Etx activity after different steps was evaluated by the minimum lethal dose (MLD) calculation, according to the standard oper-ating procedure. Toxin quantification was determined using Lowry technique, and its presence and specificity was tracked to identify pure Etx by SDS- PAGE and western blotting. Finally, the purity of Etx was evaluated by capil-lary electrophoresis.
RESULTS: The purified Etx formed a single band of about 32.9 kDa in SDS-PAGE and blotting. The pure Etx concentration was calculated to be 3.9 mg/ml and its MLD value was the dilution of 1/24000 after the ultrafiltration step. The presented purification processes to purify Etx resulted in ~ 87-fold concentration and 88.6% purity.
CONCLUSIONS: Due to this high Etx purity, the processes used in this study can provide the technical knowledge of toxin production in a larger industrial scale that can be used in development of clostridial toxoid vaccines, as well as quality control and/or diagnostic tests.
|Clostridium perfringens؛ Concentration؛ Epsilon toxin؛ High purity؛ Purification|
|عنوان مقاله [English]|
|یک روش کاربردی نوین برای تخلیص توکسین اپسیلون باکتری کلستریدیوم پرفرنژنس تیپ D، توسط کروماتوگرافی اندازه طردی (SEC) و اولترافیلتراسیون (UF)|
|مکرمه پودینه معرف1؛ محمد کاظم کوهی1؛ مجتبی علی ملایی2؛ تارا امامی3؛ جلال حسن1|
|2گروه تحقیقات و فناوری، واحد کرمان، موسسه تحقیقات واکسن و سرم سازی رازی، سازمان تحقیقات، آموزش و ترویج کشاورزی (AREEO)، کرمان، ایران|
|3گروه پروتئومیکس و بیوشیمی، موسسه تحقیقات واکسن و سرم سازی رازی، سازمان تحقیقات، آموزش و ترویج کشاورزی (AREEO)، کرج، ایران|
|زمینه مطالعه: سمیت بالقوه بالای توکسین اپسیلون تولید شده توسط باکتری کلستریدیوم پر فرنژنس ((Clostridium perfringens تیپ D، آن را به سومین سم کشنده کلستریدیال پس از بوتولینوم و کزاز تبدیل کرده است، بنابراین داشتن توکسین خالص و غلیظ اهمیت زیادی دارد. |
هدف: هدف از این مطالعه خالص سازی توکسین اپسیلون تا حد ممکن با اجرای پروتکل های کاربردی، مقرون به صرفه، چند مرحله ای و در کمترین زمان بود.
روش کار: تصفیه توکسین اپسیلون در چندین مرحله متوالی انجام شد; رسوب سولفات آمونیوم، دیالیز، کروماتوگرافی اندازه طردی با استفاده از ستون Sephadex-G50 ، دو مرحله تغلیظ و اولترافیلتراسیون. فعالیت توکسین اپسیلون پس از مراحل مختلف با توجه به روش عملیاتی استاندارد با محاسبه حداقل دوز کشنده (MLD) تعیین شد. میزان توکسین با استفاده از روش Lowry و حضور و اختصاصیت آن نیز توسط SDS-PAGE و وسترن بلات پیگیری شد. در نهایت، ارزیابی خلوص توکسین اپسیلون با اجرای الکتروفورز مویرگی صورت گرفت.
نتایج: توکسین اپسیلون خالص یک باند واحد، در حدود 32.9 کیلو دالتون در SDS-PAGE و وسترن بلات تشکیل داد. غلظت خالص توکسین اپسیلون، mg/ml3.9 محاسبه شد و میزان MLD آن، رقت 24000/1 پس از مرحله اولترافیلتراسیون بود. فرآیندهای تصفیه ارائه شده جهت تخلیص توکسین، منجر به تغلیظ آن تا fold 87 و خلوص 88.6 درصد شد.
نتیجه گیری نهایی: با توجه به خلوص بالای توکسین اپسیلون، فرایند های مورد استفاده در این مطالعه می تواند دانش فنی تولید توکسین در مقیاس صنعتی را فراهم کند که می تواند در تولید واکسن توکسوئید کلستریدیدیال، همچنین کنترل کیفیت و یا آزمایشات تشخیصی استفاده شود.
|کلستریدیوم پرفرنژنس, توکسین اپسیلون, تخلیص, تغلیظ, خلوص بالا|
Clostridium perfringens (C. perfringens) is a Gram-positive anaerobic bacterium that is not motile. It is a normal flora (i.e., microbiota) of human and animal digestive tracts, dominant in various environments, including foods, soils, dust, feces, sewage, and wastewaters (Garcia et al., 2013).
Epsilon toxin (Etx) is the most lethal toxin produced by C. perfringens type D. It is produced to a lesser extent by toxinotype B, but not other toxinotypes (Nagahama et al., 2020). Etx has a greater pathogenic potential than the other C. perfringens toxins (Garcia et al., 2013). It ranks the third most lethal clostridial toxin behind botulinum and tetanus, with an LD50 of ~110 ng/kg in mice. It is also considered a potential biological warfare and bioterrorism agent (Alves et al., 2014).
Etx is secreted as a very low-active polypeptide, the epsilon prototoxin (~33 kDa), and becomes fully active by the digestive protease enzymes like trypsin and chymotrypsin, creating the mature Etx (~29-32 kDa), and its toxicity increases up to 100 times than the prototoxin (Najafi, 2012; Zayerzadeh et al., 2015).
Since Etx is the main toxin of C. perfringens used to make enterotoxemia vaccine, having pure and concentrated Etx is very important. High purity Etx was also used for different diagnostic as serum neutralization test, immunological assays, and antibody detection in the immunized animals and/or vaccine quality control tests as potency test (Alves et al., 2021).
To date, many attempts have been made to purify Etx based on a single or a combination of several protocols. Precipitation and subsequent purification with different chromatographic techniques are the most commonly used purification procedure for Etx (Zayerzadeh et al., 2015). Ammonium sulfate precipitation is used in many studies to protein precipitation because of its simplicity and cost-effectiveness (Andler and Steinbüchel, 2017; Habeeb, 1969; Kaushik et al., 2019), albeit some disadvantages as the effect of high salt concentrations on the structure of proteins (Alves et al., 2021). Methanol precipitation was also used by Verwoerd (1960).
Chromatography is an important technique for Etx purification and is usually used as a purification step in almost all studies. The most used chromatographic methods to purify Etx are hydrophobic interaction, ion-exchange, gel filtration, and affinity chromatography. These can be used in single-step (Mathur et al., 2010) or a combination of several consecutive steps (Boarer et al., 1988; Payne et al., 1994; Zayerzadeh et al., 2015). The approach for Etx purification is ion-exchange chromatography (Habeeb, 1969). Although ion-exchange chromate-graphy provides acceptable results (Alves et al., 2021; Zayerzadeh et al., 2015), the effect of buffer type on peak resolution cannot be ignored. Also, unlike gel filtration chromatography, excessive and frequent use of columns in this method may affect the analysis results (Alves et al., 2021).
Another way for protein purification is ultrafiltration that can be performed in a lab or industrial scale. Ultrafiltration is a pressurized process using semi-permeable membranes that separates sample components based on size and response to a driving force. This separation involves the removal of large amounts of solvent from the sample and the concentration of the particles (Diaz et al., 2018).
Until today, the best purified Etx (77-fold purification) was obtained via precipitation and ion-exchange chromatography (DEAE-C) (Worthington et al., 1973). There are still defects in the chromatography purification process to obtain high pure Etx, including using two chromatographic steps as ion-exchange followed by gel filtration chromatography in the study of Alves et al. (2021), which, although increasing purity, reduces performance.
Furthermore, applying two chromatographic techniques is also not cost-effective. For these reasons, researchers prefer to utilize a well-applicable one-step or multistep purification method based on chromatography, filtration, and/or a combination of both (Alves et al., 2021; Worthington et al., 1973). The most important bottlenecks faced by researchers in choosing the best purification procedure are low mobile phase volume, cost-saving, sample structure stability, simplicity, good sensitivity, etc. (Ó’Fágáin et al., 2017). The aim of this study was to purify Etx as pure as possible with an applicable, cost-effective, multistep purification protocol with the lowest and shortest time.
Materials and Methods
All experimental manipulations were performed according to the local institutional guidelines and standard operating producers (SOPs) for the care and use of laboratory animals, Institute of Razi, Iran. This study was carried out under the 3Rs (replacement, reduction, and refinement) principles, and efforts were made to minimize animal suffering.
C. perfringens Culture and Toxin Preparation
The toxic filtrates used were obtained from 1 liter of culture C. perfringens type D. The purification of the Etx was carried out in multiple stages; precipitation, dialysis, size exclusion chromatography (gel filtration by G50), concentration, and ultrafiltration.
Following the culture of the bacterium, the cell debris was separated from the supernatant using centrifugation (6000 rpm for 40 minutes at 4°C). Proteins in supernatant were precipitated by adding 500g of 50% ammonium sulfate (AM04011000, Scharlau, Spain) in two steps, stirring at room temperature for 2 h, and stored at 4°C for 24 h (Payne et al., 1994). Then, the pellet was collected after centrifugation (6000 rpm for 40 minutes at 4°C), resuspended in Tris-HCl (0.02 M, pH 6.3), and dialyzed by dialysis tubing MWCO 10kDa (Cat Number 71748-3, Millipore) to remove the salt. Dialysis was performed against the same buffer for 4 h at 4°C and the sample was collected at the end and kept at -20°C for further purification.
Following dialysis, the Etx was purified by size exclusion chromatography (gel filtration with G50). Sephadex G-50 Fine powder (9048-71-9, Sigma) was dissolved in Tris-HCl buffer (0.02 M, pH 6.3) and packed into the chromatography column (2 x 55 cm). The concentrated sample was loaded on the prepared column, and the flow rate was set by the peristaltic pump to 30 mL/hour. Then the column was eluted by the same buffer, and the output fractions (3 mL per tube) were collected. The fractions' optical absorption at 280 nm was measured, and peaks with high Etx concentration were selected and pooled to obtain more pure toxin.
The pooled sample passed through a 0.45 μm syringe filter (CHROMAFIL CA-45/25 S, 729025, Macherey-Nagel) to remove particulate impurities and sample filtration. Then, Etx was concentrated in two steps by a dialysis tubing cellulose membrane (MWCO 10 KDa, D9402, Sigma) with sucrose crystals around it to allow water to flow out for 1 hour. This procedure should be continued until the sample volume is halved (Noman et al., 2006). Next, the concentrated Etx were collected and stored at -20°C.
This procedure is performed to eliminate the smaller proteins and improve Etx purity. Amicon® Ultra-4 Centrifugal Filter Unit (NMWL: 30kDa, UFC803008, Millipore) was used since the molecular weight of Etx is around 32 to 34 kDa. The sample was loaded into the tube and centrifuged for 45 min-utes at 5000 rpm at 4°C (Boarer et al., 1988). The ultra-pure Etx was stored at -70°C for further analysis.
Evaluation of Etx Purification
The presence of Etx at different purification steps was confirmed by calculation of the MLD at BALB/c mice (weight 20±2 g, 6-8 weeks). Samples obtained by different purification steps, including precipitation, chromatography, and ultrafiltration, were first activated by trypsin enzyme (500 µL toxin + 5 mg trypsin), then different dilutions were prepared and injected in tail vein (2 mice per dilution). Finally, mice were followed for 72 hours later to estimate toxin lethality. Toxin quantification was also evaluated using the technique of Lowry et al. (1951).
Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) method was used to track Etx in unpurified samples and/or to identify pure Etx. SDS-PAGE was performed using the Laemmli method (1970). The coomassie brilliant blue R-250 was used to stain the bands of the chromatographic fractions, and silver nitrate was used to stain the sample bands concentrated by ultrafiltration and the pooled fractions following chromatography (Hochstrasser et al., 1988). The specificity of pure Etx was evaluated with 5 IU/mL of the specific sheep Anti-epsilon HP serum (RVSRI, Iran) as the primary antibody and 0.2 µg/mL of the rabbit poly anti-sheep IgG (H&L), HRP (KO211798, Koma Biotech Inc.) as the secondary conjugate antibody by western blotting, according to the standard protocol (Sayeed et al., 2005). Bands were detected using DAB substrate, and specific bands of the purified Etx were observed.
The purity of Etx was evaluated by capillary electrophoresis using the Agilent 7100 CE (Agilent Technologies, USA), with an ultraviolent- visible (UV–vis) detector at 200 nm. For this work, uncoated fused silica capillaries (50 m ID x 360 m OD x 70 cm length) were utilized. Prior to loading the sample, 100 µL of pure Etx was mixed with 1900 µL of 10 mM acetic acid, and this prepared sample was then analyzed using Agilent ChemStation software.
The primary culture of C. perfringens type D was performed in a specific medium. The Etx expression was improved by consecutive cultures of the bacterium with increasing the medium volume and subsequently in a toxin-producing medium in the fermenter under controlled pH and temperature. In all stages, there was good growth. The purity of cultures was controlled and confirmed by Gram staining smears. The presence of Etx in the supernatant of one liter of culture medium in the fermenter was confirmed by IV injection of 0.5 mL of the supernatant solution to two mice. Death of animals in this group compared to the control group (normal saline recipient) showed the activity of Etx. Toxin activity was estimated by MLD assay, which was 1/8000.
Precipitation and Gel Filtration Chromatography
The final volume of solution after precipitation and concentrated dialysis was 30 mL and 20 mL, respectively. After these primary precipitation and purification steps, the total protein concentration and MLD value were estimated at 50 mg /mL and 1/14000, respectively.
After precipitation and dialysis, column chromatography (Sephadex G-50 gel filtration) was used to purify Etx. The output fractions were collected and evaluated for 280 nm optical absorption of each by spectrophotometer, and a chromatogram was plotted (Figure 1).
The protein concentration of chromatographic fractions was measured by the Lowry method at a wavelength of 750 nm, and their proteins banding pattern was examined by vertical electrophoresis of polyacrylamide gel (SDS-PAGE) (Figure 2). According to the electrophoresis results, the fractions containing pure Etx were mixed to obtain a higher toxin concentration and were used for the next purification step. The protein content of the pooled fractions was estimated at about 2.1 mg/mL, and its MLD was determined by the dilution of 1/19000.
The purification of culture supernatant by ammonium sulfate precipitation and dialysis, followed by Sephadex G-50 filtration chromatography, resulted in ~ six-fold and ~13-fold concentration of the Etx, respectively.
Figure 1. Chromatogram of gel filtration chromatography with Sephadex G-50
Figure 2. Vertical polyacrylamide gel electrophoresis (SDS-PAGE) on some of fractions after column chromatography with Sephadex G-50 gel and coomassie blue staining. Lane L: The protein size marker; Lane 1: The crude culture supernatant containing Etx after precipitation, prior to chromatography; Lanes 1-8: Some of the fractions after chromatography that show the relative purified Etx. Note the purified Etx in lanes 4-8, which weighing about 32.9 kDa and indicated by the arrow.
Concentration and Ultrafiltration
The purified Etx was concentrated by two dialysis steps, and its purity was improved by ultrafiltration. The volume of the toxin-containing solution was 8 mL and 3.5 mL after the first and second concentration steps, respectively. The final volume of purified Etx after the ultrafiltration step was measured 1.5 mL. The protein concentrations were calculated to be 2.4 mg/mL and 3.9 mg/mL after the concentration and the ultrafiltration steps, respectively. The activity of the purified Etx after ultrafiltration was also determined by measuring the MLD in different dilutions, and the MLD value was the dilution of 1/24000.
The evaluation of the purified Etx showed an excellent purification after ultrafiltration than G-50 chromatography by SDS-PAGE (Figure 3) and western blotting (Figure 4). Purified Etx appeared as an approximately 33 kDa protein on SDS-PAGE and western blot using sheep anti-epsilon HP serum as primary antibody and anti-sheep IgG (H&L), HRP conjugated secondary antibody. The electrophoretic pattern indicates excellent purification of Etx and its high purity after ultrafiltration. The purity of the purified Etx after ultrafiltration was also measured by capillary electrophoresis (Figure 5), which is the highest peak related to the purified Etx with 88.6% purity. The final purification of Etx by ultrafiltration resulted in ~ 87-fold concentration. The purification process of C. perfringens Etx was summarized in Table 1.
Figure 3. The vertical polyacrylamide gel electrophoresis (SDS-PAGE) of pooled fractions containing purified Etx after chromatography with Sephadex G-50 and ultrafiltration with Amicon® Ultra-4 Centrifugal Filter staining with the silver nitrate. Lane L: The protein size marker; Lane 1: The crude culture supernatant containing Etx after precipitation, prior to chromatography; Lane 2: High pure Etx after ultrafiltration; Lane 3: The purified Etx after G50 column chromatography. The pure Etx weighing about 32.9 kDa is indicated by arrow.
Figure 4. Western blot analysis of the purified Etx after chromatography and ultrafiltration. Lane L: The protein size marker; Lane 1: The purified Etx after G50 column chromatography; Lane 2: High pure Etx after ultrafiltration. The pure Etx weighing is about 32.9 kDa
Figure 5. Graph of the purity of the purified Etx by capillary electrophoresis technique. The highest peak based on the relative passage time of soluble components from the detector is related to the purified Etx with 88.6% of purity.
Table 1. Summary of the purification process of the Etx from C. perfringens type D strain CN409
a Specific activity (MLD/mg) = Activity (MLDs)/ mg of total protein
b Purification (fold) = final specific activity / initial specific activity
c Yield (%) = (final Total Toxin / initial Total Toxin) × 100
d Efficiency = (Purification (fold) × Yield (%))/100
The high potential toxicity of Etx produced by C. perfringens type D, has made it a very important toxin among other clostridial toxins (Alves et al., 2014; Garcia et al., 2013). It is responsible for the pathogenesis of pulpy kidney disease or enterotoxemia. The production of purified Etx has many applications as quality control tests, serum neutralization, and antibody detection in vaccinated animals (Alves et al., 2021; Mathur et al., 2010), and its industrial purification by ultrafiltration can be used for toxoid vaccine production. Intending to increase Etx purification, the current study focused on the procedures of precipitation, chromatography, concent-ration, and ultrafiltration, eventually reaching a purity of 88.6% of the Etx.
The best strategy used to purify toxins is to use a cost-effective, simple, relatively fast method that yields the maximum purity (Garcia et al., 2013; Morcrette et al., 2019). Ammonium sulfate precipitation and chromatography techniques are the most commonly used purification procedure for Etx (Kaushik et al., 2019; Najafi, 2012; Zayerzadeh et al., 2015). The optimum concentrations of ammonium sulfate for the Etx precipitation are reported to be 50-70% (Kaushik et al., 2019; Zayerzadeh et al., 2015). In this study, a concentration of 50% ammonium sulfate was used due to the sensitivity and high degra-dability of Etx, as high concentrations of ammonium sulfate can lead to its degradation (Alves et al., 2021). The amount of total protein after precipitation (50 mg) indicates that the concentration of ammonium sulfate is appropriate.
Various chromatographic techniques are commonly used for Etx purification in almost all studies (Singh et al., 2020; Zayerzadeh et al., 2015). In some studies, two chromatographic techniques were utilized to improve the purity of Etx, which, in addition to decreasing yield, increases the danger of protein breakdown and reduces its effectiveness. For example, it was discovered that the use of ion-exchange and gel filtration chromatography, as well as anion exchange repeat chromatography, did not provide any obvious advantage compared to single-step chromatography (Alves et al., 2021). This result was observed in the study of Zayerzadeh et al. (2015), which also used cation exchange chromatography followed by gel filtration, and in Habib's research (1969), which applied two ion-exchange chromatography steps. Therefore, it seems that using a chromatographic step not only saves time and money but also provides superior outcomes in lab-scale purification. In the present study, single-step gel filtration chromatography was used. Following that, the fractions with the highest concentration of Etx were pooled together based on the SDS-PAGE result to obtain a higher concentration of Etx.
An important advantage of gel filtration is that the parameters may be adjusted without affecting the separation depending on the sample or the requirement for additional purification, analysis, or storage conditions (Ó’Fágáin et al., 2017). Because molecules do not interact with the chromatographic medium in the same way, the buffer content has no direct influence on the degree of separation between the peaks. This is probably why the purification factor in the study of Worthington et al. (1973), who used anion-exchange chromatography step after precipitation with ammonium sulfate, was 77-fold), whereas in this study and following gel filtration and ultrafiltration, and the Etx was purified 87-fold. Capillary electrophoresis results indicate this level of purity.
In the present study, performing a single-step chromatography and an additional ultrafiltration step, the final yield of Etx was 20.31%. In the study of Zayerzadeh et al. (2015), despite one more ion-exchange chromatography step, the final yield was 16.7%. On the other hand, because the chromatography was performed in one step, the toxin degradation was largely prevented following a reduction in the purification time. The type of usage and the final objective of purification determined how each purification procedure is implemented (Alves et al., 2021). After anionic chromatography and ultrafiltration, the final yield in the Boarer et al. (1988) study was reported to be 0.331 mg/mL (Boarer et al., 1988), while in the present study, it was 3.9 mg/mL, which indicates the high efficiency of the method used. As a result, it seems that in similar conditions, performing gel filtration chroma-tography compared to ion-exchange chromatography greatly increases the yield of the Etx.
According to previous studies, the inactive form (protoxin) of Etx weighs around 33 kDa, whereas the fully active form weighs around 29 kDa (Bokori et al., 2011; Payne and Oyston, 1997). Based on the current gel electrophoresis results, the molecular weight of the purified Etx in this study was about 32.9 kDa, which corresponded to the molecular weight of the studies using different chromatographic methods (Nagahama et al., 2020; Najafi, 2012; Payne et al., 1994), so the type of purification method has no significant effect on the molecular weight of the resulting pure Etx.
The ultrafiltration method was used to concentrate and purify Etx. This method, which is based on the separation of compounds based on particle size, allows smaller particles to pass while particles larger than the membrane pores are preserved (Diaz et al., 2018). Since the molecular weight of Etx is between 32 and 34 kDa (Najafi, 2012; Zayerzadeh et al., 2015), in this study 30 kDa ultrafiltration membrane was used. In most of the previous studies (Alves et al., 2021; Payne et al., 1994; Zayerzadeh et al., 2015), preparation of pure toxin was considered, while in the current study, as pure as possible, in addition to the high purity of the toxin (which is the main goal), the attainment of a concentrated toxin is also considered. Therefore, it is necessary to concentrate toxin after the purification steps to obtain a higher amount of pure Etx.
This study will provide technical knowledge on the production and purification of Etx with high concentration from type D C. perfringens. This pure Etx in industrial production scale by ultrafiltration systems can be used to develop clostridial toxoid vaccines, as well as quality control and/or diagnostic tests.
This work was supported by the Razi Vaccine and Serum Research Institute of Iran under Grant [number 12-18-18-066-96029-960766]. We would like to thank the workers of Proteomics and Biochemistry, and Anaerobic Bacterial Vaccine Rese-arch and Production Departments of the Razi Vaccine and Serum Research Institute for their help and support.
Conflict of Interest
The author declares no conflict of interest.
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