Purification, characterization and immunostimulati

Ye Seul Na a ,b ,Woo Jung Kim a ,b ,Sung Min Kim a ,b ,Jae Kweon Park a ,b ,Sae Mi Lee a ,b ,Sung Oog Kim a ,b ,Andriy Synytsya c ,Yong Il Park a ,b ,⁎

a Department of Biotechnology,The Catholic University of Korea,Bucheon,Gyeonggi-do 420-743,Republic of Korea

b Biomaterial Engineering Research Center,The Catholi

c University of Korea,Bucheon,Gyeonggi-do 420-743,Republic of Korea

c

Department of Carbohydrate Chemistry and Technology,Institute of Chemical Technology in Prague,Technická5,16628Prague 6,Czech Republic

a b s t r a c t

a r t i c l e i n f o Article history:

Received 4November 2009

Received in revised form 17December 2009Accepted 27December 2009Keywords:

Sulfated polysaccharide Capsosiphon fulvescens Immunostimulation Macrophages

A water-soluble polysaccharide (SPS-CF)was isolated and puri fied from Korean Capsosiphon fulvescens by dilute acid extraction,ethanol precipitation,and DEAE-cellulose ion exchange chromatography.The puri fied SPS-CF was shown to be a glucuronogalactomannan with a molecular mass of 385kDa and the monosaccharide composition of the SPS-CF was determined to be mannose (55.4%in mole percentage),galactose (25.3%),glucuronic acid (16.3%),and arabinose (0.8%).Fourier-transform infrared and elemental analysis indicated that the puri fied SPS-CF is a sulfated polysaccharide containing signi ficant amount of sulfate esters (5.7%in mass).Enzyme Linked Immunosorbent Assay showed that the SPS-CF signi ficantly stimulates the release of the pro-in flammatory cytokines,TNF-αand IL-6,in a dose-dependent manner.RT-PCR analysis demonstrated that the SPS-CF also induced a more than two-fold increase in the expression of iNOS and COX-2,responsible for the induction of NO and PGE2,respectively,at 5µg/ml in RAW2.7murine macrophages.These results suggest that the sulfated SPS-CF isolated from C.fulvescens has potent immunostimulating activity.

©2010Elsevier B.V.All rights reserved.

1.Introduction

Sulfated polysaccharides (SPS)that contain hemi-ester sulfate groups in their sugar residues are commonly found in three major groups of marine algae,red algae (Rhodophyta),brown algae (Phaeophyta)and green algae (Chlorophyta)[1,2].The major SPS of red algae are galactans commercially known as agar and carrageenan,and those of brown algae are fucans,including fucoidan,ascophyllan,sargassan,and glucuronoxylofucan [1].In contrast to those of red and green algae,the major SPS of green algae are more heterogeneous in sugar compositions,three main groups are glucuronoxylorhamnans,glucuronoxylorhamnogalactans and xyloarabinogalactans [1].Apart from industrial uses,these algal polysaccharides have emerged in recent years as a rich and important source of bioactive natural com-pounds.For example,fucoidans,one of the most extensively studied SPS,are known to exhibit a wide range of physiological and biological activities,thus medically useful activities,such as anti-in flammatory [3],antiviral [4],anticoagulant [1,5–9],antitumor [10,11],antioxidant [12],and antiangiogenesis [13]activities.For these reasons,production and applications of these acidic polysaccharides as therapeutic agents have been increasingly important topics of intensive researches.

Due to several side effects of heparin,anticoagulating and anti-thrombotic activities are among the most widely studied properties of SPS for alternative sources of heparinoid-active compounds as therapeutic agents.Comparing to the red and brown algal SPS,reports on the biological activities of green algal SPS are relatively recent and fewer.Some SPS of green algal species have been reported for their anticoagulating and antithrombotic [14–18],antitumor [2],and immunomodulating [19]activities.

Capsosiphon fulvescens ,a green alga,grows in copious amounts along the southwestern coast of Korea and also widely throughout the world,including European and North American coasts [8].Although the C.fulvescens has been traditionally used in Korea as a functional food for centuries,the uses and potential values of bioactive materials from this species have not been well studied.Accordingly,in an attempt to search for such compounds,this study reports the puri-fication and chemical characterization of a sulfated polysaccharide (SPS-CF)from Korean C.fulvescens and its immunostimulating effects on murine RAW2.7cells.2.Materials and methods 2.1.Extraction of polysaccharide

C.fulvescens was collected from the southern coastal area of Wando,Korea.The extraction of polysaccharide was performed as described by Kim et al.(2007)with minor modi fications [6].Brie fly,

International Immunopharmacology 10(2010)3–370

⁎Corresponding author.Department of Biotechnology,The Catholic University of Korea,Bucheon,Gyeonggi-do 420-743,Republic of Korea.Tel.:+822214512;fax:+822214846.

E-mail address:yongil382@catholic.ac.kr (Y.I.

Park).1567-5769/$–see front matter ©2010Elsevier B.V.All rights reserved.doi:

10.1016/j.intimp.2009.12.011

Contents lists available at ScienceDirect

International Immunopharmacology

j o u r na l h o m e p a g e :w w w.e l s ev i e r.c o m /l o c a t e /i n t i m p

2.2.Purification of polysaccharide

The crude polysaccharide,named SPS-CF,was further purified by column chromatography.Fifty milligrams of crude polysaccharide dissolved in10ml of dH2O was applied to a DEAE-cellulose column (3×45cm)pre-equilibrated with water and eluted in NaCl gradient (0–3M)until no carbohydrate is detected.Each fraction was assayed for carbohydrates content by phenol-sulfuric acid method[20].The carbohydrate-positive fractions were pooled together and dialyzed (MWCO14,000)for24h against dH2O and lyophilized.

2.3.Chemical analysis

The total carbohydrate of the SPS-CF was determined by the phenol-sulfuric acid method at490nm[20].The amount of sulfate residues was determined by Loui's method[21],using Na2SO4as a standard.Uronic acid content was determined by the carbazole reaction[22],using D-glucuronic acid as a standard.Protein was quantified by Bradford method[23].

2.4.Monosaccharide composition analysis

The monosaccharide composition of the purified SPS-CF was analyzed by HPLC after2M trifluoroacetic acid(TFA)hydrolysis and labeling of the hydrolysate with1-phenyl-3-methyl-5-pyrazolone (PMP,Sigma).The resulting PMP-labeled monosaccharides were ana-lyzed on a Shim-Pack CLC-ODS column(6.0mm×15cm,Shimadzu, Japan)using an HPLC(P680,Dionex,USA)system equipped with a UV detector(UVD170U,Dionex,USA)at aflow rate of1ml/min in a gradient mode with buffers A(0.1M sodium phosphate buffer,pH7.0, containing10%CH3CN)and B(same buffer with25%CH3CN). Monosaccharides were identified and quantified by comparing to authentic ones used as standard.

2.5.Elemental analysis

Elemental analysis on the carbon,hydrogen,nitrogen and sulfur weight percentages of SPS-CF was performed by using the EA1112(CE Instrument,Italy)using a CHNS-Porapack PQS(CE Instrument,Italy). Approximately1mg of sample was injected,eluted with He and detected with TCD(Thermal conductivity detector,CE Instrument). 2,5-Bis(5′tert-butyl-2-benzoxazol-2-yl)thiophene(BBOT)and as-partic acid were used as a reference standard.

2.6.Estimation of the molecular weight

The average of the relative molecular weight of the polysaccharide was estimated by size-exclusion HPLC(Dionex,USA)using a Shodex OHpak column(SB-806HQ,8.0×300mm,Showa Denko Co.,Japan). Ten microliter of1%of the SPS-CF(in water)was injected,eluted with water at aflow rate of0.8ml/min at60°C and detected with ELSD (Evaporative light scattering detector,Alltech).Dextrans were used as the relative molecular mass standards:162,000,143,000,71,300,and 43,000Da(Sigma,USA).

2.7.Vibration spectroscopy

Fourier-transform infrared(FTIR)spectrum(spectral region4000–400cm−1,and resolution4cm−1)of the polysaccharide sample was recorded on Nicolet Magna550series II(Midac,USA)spectrometer using the attenuated total reflection(ATR)technique of measuring. FT Raman spectrum was recorded by using a Bruker FT Raman (FRA106/S,Equinox55/S)spectrometer equipped with a quartz beam splitter,a liquid nitrogen cooled germanium detector and excitation at10nm from a Nd:YAG laser.The laser power was set at100mW, and256scans were accumulated with a spectral resolution of2.0cm−1. The position of overlapped bands was obtained with an assistance of the2nd derivative algorithm.Vibration spectra were smoothed and baseline corrected using Origin6.0(Microcal Origin)software.

2.8.MTT cytotoxicity assay

Briefly,the toxicity profiles of SPS-CF were assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide(MTT) microculture tetrazolium viability assay[24].Precultured RAW2.7 cells(5×104cells/well)in RPMI10media were plated on a96-well microplate.Different concentrations of the SPS-CF were prepared with serial dilutions ranging from10to1.25µg/ml.After24h of incubation, 20µl of MTT(5mg/ml)was added to each well and the plates were incubated for4h and the media removed.Two hundred microliter of DMSO was later added into each well to solubilize the formazan crystals. The absorbance was read at595nm using a microplate reader.

2.9.Assay for cytokine secretion

The concentration of tumor necrosis factor-α(TNF-α),interleukin-6 (IL-6)and prostaglandin E2(PGE2)released from murine RAW2.7 cell line treated with the purified SPS-CF was determined,using an ELISA assay(Quantikine Immunoassay Mouse TNF-α,Quantikine Immuno-assay Mouse IL-6,R&D systems,USA,Prostaglandin E2EIA Kit,Cayman, USA).RAW2.7cells(5×104cells/ml)subcultured on a96-well mic-roplate in RPMI10media were incubated for24h in the presence of various concentrations of the purified SPS-CF and lipopolysaccharide (LPS)as test compound.LPS used in this study was Escherichia coli EH100endotoxin(Sigma).Supernatant from each well was harvested and cytokine concentration was measured by optical density following the manufacturer's instructions.

Table1

Chemical composition and molecular weight of the SPS-CF.

Neutral

sugar a

(mass%)

Sulfate b

(mass%)

Uronic

acid c

(mass%)

Protein d

(mass%)

Proportion of

monosaccharides e

(mole%)

Molecular

weight f

(kDa)

Man Gal GlcU Ara

SPS-CF49.8 5.7 4.8055.425.316.30.8385

a Neutral sugar was determined by phenol-sulfuric acid method[20]at490nm.

b The amount of sulfate residues was determined by Loui's method[21].

c Uronic aci

d content was determined by th

e carbazole reaction[22],using D-glucuronic acid as a standard.

d Protein was quantified by Bradford method[23].

e Values were obtained by setting the sum o

f each mole number at100%.

f The molecular weight of purified polysaccharide was estimated by size-exclusion HPLC usin

g dextrans as standard size markers(Sigma):162(a),143(b),71.3(c), 43kDa(d).

365

Y.S.Na et al./International Immunopharmacology10(2010)3–3702.10.Reverse transcriptase-polymerase chain reaction(RT-PCR)

To evaluate the mRNA expression levels of inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2),the total RNA from LPS and SPS-CF treated(or untreated)RAW2.7cells was prepared by using a Total RNA extraction kit(Intron Biotechnology,Korea).The total RNA was stored at−80o C for further use.The RT-PCR was performed using the ONE-STEP RT-PCR PreMix kit(Intron Biotech-nology,Korea).The products of the RT-PCR were separated by elec-trophoresis using2%agarose gel stained with ethidium bromide,and the gels were viewed under UV transillumination.The primers used in this experiment are indicated in Table1(F,forward;R,reverse).

3.Results

3.1.Extraction and purification of polysaccharide

Polysaccharide was extracted from the C.fulvescens by dilute acid treatment and the crude preparation was obtained following75% ethanol precipitation and CaCl2treatment,with the yield of approx-imately2.49%of the dry mass of C.fulvescens.As shown in Fig.1,DEAE-cellulose column chromatography showed two major carbohydrate-positive peaks.Fractions of170–220were combined,concentrated and designated as the SPS-CF fraction.The polysaccharide fraction was eluted at approximately1.3M NaCl in water.

3.2.Chemical composition of the SPS-CF

Chemical composition of the purified polysaccharide(SPS-CF)was determined as shown in Table1.The SPS-CF was composed of neutral sugars(49.8%),sulfate(5.7%),uronic acid(4.8%)and protein(0%in mass).In addition,four elements of the purified SPS-CF were deter-mined to be:carbon(31.9%w/w),hydrogen(4.9%w/w),nitrogen (0.6%w/w)and sulfur(6.2%w/w)as summarized in Table2.Taken together,the results clearly demonstrated the presence of sulfate groups in this polysaccharide.The monosaccharide composition of the purified SPS-CF was determined to be mannose(55.4%),galactose (25.3%),glucuronic acid(16.3%),and arabinose(0.8%)in molar per-centage,based on the HPAEC-PAD analysis(Table1and Fig.2).These results suggest that the SPS-CF is a heteropolysaccharide,a galactomannan mainly consisting of galactose and mannose.The apparent average molecular weight of the purified polysaccharide was estimated to be approximately385kDa by size-exclusion HPLC (Table1).

3.3.Vibration spectra of SPS-CF

FTIR(ATR technique)and FT Raman spectra of this sample are demonstrated in Figs.3and4.Band assignment was made according to the literature[25–27].Broad IR band centered at3410cm−1arises from OH stretching vibrations of hydroxyls and water.Moderate IR bands at1620cm−1and1416cm−1arises from asymmetric and symmetric stretching vibrations of carboxylate[28,29].Raman band of the symmetric vibration of COO−was found at1413cm−1.Intense IR band at1223cm−1and very intense Raman band at1180cm−1 were assigned respectively to asymmetric and symmetric stretching vibrations of O S O that is an evidence of sulfate esters[25].Strong highly overlapped IR/Raman bands in the region of900–1200cm−1 are characteristic for carbohydrates and correspond to coupled CO and CC stretching and COH bending vibrations in polysaccharides. IR/Raman band at9cm−1is characteristic forβ-polysaccharides. Therefore,according to FTIR and FT Raman band assignments the purified sample is sulfated polysaccharide of complex structure con-taining uronic acids as a minor sugar

component. Fig.1.Fractionation of crude polysaccharide by DEAE-Cellulose column chromatography.

Table2

Elemental analysis of SPS-CF.

Element Theological composition

(%w/w)Experimental composition (%w/w)

BBOT Aspartic acid SPS-CF a

%N 6.5110.520.6318

%C72.5336.0931.9398

%H 6.09 5.30 4.9022

%S7.44– 6.2166

a Approximately1mg of SPS-CF was analyzed.2,5-Bis(5′tert-butyl-2-benzoxazol-2-yl)

thiophene(BBOT)and aspartic acid were used as the reference standards.

366Y.S.Na et al./International Immunopharmacology10(2010)3–370

3.4.Macrophage activation by the SPS-CF

RAW2.7cells are known to produce cytokines in response to the addition of LPS,and this system is used to detect the modulat-ing activities of compounds on cytokine production.The defense mechanism of macrophages against pathogens includes pro-in flam-matory cytokines such as TNF-α,IL-1,IL-6,and others.The SPS-CF (10µg/ml)stimulated the production of TNF-αfrom macrophages (5×104cells/ml)to a level 80-times greater than the normal (Fig.5A).Also the SPS-CF apparently induced IL-6secretion from RAW2.7

cells in a dose-dependent manner (Fig.5B).These results suggest that the puri fied SPS-CF has bene ficial effects on immunostimulation (Table 3).

3.5.Activation of iNOS and COX-2

Expression of iNOS and COX-2in macrophages is largely regulated by transcriptional activation.It has been implicated that activated macrophages also release some immune factors including iNOS,COX-2,and others.To assess the macrophage activation by the puri fi

ed

Fig.2.HPLC analysis for monosaccharide composition of the puri fied polysaccharide.Man,Mannose;GlcU,glucuronic acid;Rha,rhamnose;Glc,glucose;Gal,galactose;Xyl,xylose;Ara,arabinose;Fuc,fucose (Sigma,

USA).

Fig.3.FTIR spectrum of the puri fied polysaccharide from the Capsosiphon fulvescens .

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Y.S.Na et al./International Immunopharmacology 10(2010)3–370

SPS-CF,RT-PCR analysis was performed to evaluate the expression of iNOS and COX-2mRNA from RAW2.7cell line (Fig.6A).The levels of mRNA expression of iNOS and COX-2increased in a SPS-CF dose-dependent manner,which were comparable to the levels with the LPS treatment.As estimated by densitometric measurement,10µg/ml of the SPS-CF stimulated the expression of iNOS and COX-2from mac-rophages (5×104cells/ml)twice greater than the normal (Fig.6B).And expression of iNOS was found to be statistically signi ficant above 2.5µg/ml.This level was almost the same as that obtained with 0.1µg/ml LPS treatment.In addition,the SPS-CF also induced pro-duction of PGE2.Stimulation of PGE2production was further con-firmed by ELISA assay (Fig.7).SPS-CF gradually increased PGE2production up to 180pg/ml at 10µg/ml,indicating an increased expression of COX-2enzyme.4.Discussion

Marine algae are an abundant source of new bioactive natural compounds with a wide range of important biological activities,such as anticoagulating and antithrombotic activities [1].Searching for new biomaterials which can potentiate the immune function has become an important area of immunopharmacological and oncotherapeutical researches.C.fulvescens has been traditionally used in Korea as a functional food for centuries with its unique flavor and soft texture and also often used to treat stomach disorders and hangovers.In this context,we isolated and characterized a water-soluble polysaccharide from Korean C.fulvescens and examined its immune stimulatory ac-tivity.The polysaccharide,SPS-CF,puri fied by the procedure described in the present study accounts for 2.49%of the dry mass of C.fulvescens .Monosaccharide composition of the puri fied polysaccharide

(SPS-CF)

Fig.4.FT Raman spectrum of the puri fied polysaccharide from the Capsosiphon fulvescens

.

Fig.5.Immunomodulating effects of puri fied polysaccharides extracted from Capsosi-phon fulvescens .RAW2.7cells were co-incubated with the indicated doses of SPS-CF for 24h.As a control,commercial LPS (0.1μg/ml)was used.The level of TNF-α(A)and IL-6(B)in the supernatants of the cultures was determined by ELISA kit.The cells were treated with LPS (0.1μg/ml)or different concentrations (1,5and 10μg/ml)of the puri fied SPS-CF.

Table 3

The primer sequences of the investigated genes in an RT-PCR analysis.Gene

Primer sequences

GAPDH F 5′-CATTTTCTTCTCCTGCAGCC-3′R 5′-TCTCCATGGTGGTGAAGACA-3′

iNOS F 5′-CTGCAGCACTTGGATCAGGAACCTG-3′R 5′-GGGAGTAGCCTGTGTGCACCTGGAA-3′COX-2

F 5′-CCCCCACAGTCAAAGACACT-3′R 5′-GAGTCCATGTTCCAGGAGGA-3′

368Y.S.Na et al./International Immunopharmacology 10(2010)3–370

was mannose (55.4%in mole percentage),galactose (25.3%),glucuronic acid (16.3%),and arabinose (0.8%),indicating that it is a heteropoly-saccharide,glucuronogalactomannan (Table 1and Fig.2).Its relative molecular mass was determined to be approximately 385kDa.Previously,Kwon and Nam [2]prepared polysaccharides (SPS-CF)having antitumor activity from Korean C.fulvescens mainly by extraction

with water at 80°C and then ethanol precipitation [2].The sugar composition of this ethanol-precipitated preparation was xylose (85%)and mannose (15%),which is quite different from our SPS-CF.This difference in component sugars may have arisen mainly from the differences in isolation and puri fication procedures or possibly the two are totally different polysaccharides present in the same C.fulvescens species.The sugar composition of our SPS-CF was also different from those of polysaccharides (glucuronoxylorhamnans,glucuronoxylor-hamnogalactans and xyloarabinogalactans)isolated from the other Chlorophyta species [1].The FTIR,Raman and elemental analysis clearly demonstrated that our polysaccharide contains a signi ficant amount of sulfate esters (5.7%in mass),indicating that it is a sulfated glucuronogalactomannan,thus designated as SPS-CF (Figs.3and 4and Table 2).Many green algal SPS,those mentioned above,are known to be covalently attached to the protein (characterized as proteoglycans)[1,30].However,the FTIR,Raman,elemental analysis and Bradford protein assay clearly showed no or negligible level of nitrogenous compound in our SPS-CF,suggesting that it does not contain protein (Figs.3and 4and Table 2).

Enzyme Linked Immunosorbent Assay demonstrated that the SPS-CF signi ficantly stimulates the release of the pro-in flammatory cytokines,TNF-αand IL-6,in a dose-dependent manner in RAW2.7murine macrophages at 1–10µg/ml,which are comparable to the levels with the LPS treatment (Fig.5A and B).Macrophages play an important role in regulating innate immunity as well as adaptive immune responses by production of cytokines such as interleukin-1β(IL-1β),IL-6,TNF-αand interferon-γ(INF-γ),and various types of chemokines such as RANTES,MCP-1,MIP-1α,TARC,etc.[31–34].Cytokines are signaling molecules that control homeostasis of

the

Fig.6.Effects of the puri fied SPS-CF and LPS on the expression of cytokines in RAW2.7cells.Cells (5×104cells/ml)were incubated with the puri fied SPS-CF (1.25,2.5,5and 10μg/ml)or LPS (0.1μg/ml)for 24h.(A)The mRNA levels were determined by RT-PCR.(B)The amount of iNOS and COX-2mRNA,expressed as the ratio of densitometric measurement of the sample to the corresponding internal standard (GAPDH),is shown in the lower

panel.

Fig.7.Effects of the puri fied SPS-CF on the induction of PGE2.RAW2.7cells were co-incubated with the indicated doses of the puri fied SPS-CF for 24h.As a control,commercial LPS (0.1μg/ml)was used.The level of PGE 2in the supernatants of the cultures was determined by ELISA kit.The cells were treated with LPS (0.1μg/ml)or different concentrations (1.25,2.5,5and 10μg/ml)of the puri fied SPS-CF.

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organism by the regulation of cell differentiation,proliferation and apoptosis,as well as defense functions such as immune responses and inflammatory reactions.TNF-αis involved in systemic inflam-mation and stimulates the acute phase reaction.IL-6is also consi-dered as an important mediator of acute inflammatory responses and functions as a differentiation and growth factor of hematopoietic precursor cells,B cells,T cells,keratinocytes,neuronal cells,osteoclasts, and endothelial cells[35].Many algal polysaccharides have been re-ported for their immunomodulatory activities in mammals as they modify the activity of macrophages.Polysaccharides from the red alga, Porphyra yezoensis[36,37]and Gracilaria verrucosa[38],showed macrophage-stimulating activity in vitro and in vivo.Carrageenan,an SPS obtained from red algae,primes mice leukocytes to produce TNF-αin response to bacterial LPS[39].Ulvan,a sulfated glucuronorhamnan from the green algae Ulva rigida,stimulates macrophage to secrete PGE2 and induce an increase in COX-2and NOS-2expression[19].Similarly, our study demonstrated that our SPS-CF also dose-dependently stim-ulates the expression of iNOS and COX-2genes,responsible for the induction of nitric oxide(NO)and PGE2,respectively,in RAW2.7 macrophages(Fig.6).NO and PGs are produced as result of the stimulation of iNOS and COX-2,respectively.NO acts as an intracellular messenger and regulates cellular functions,such as inflammation,as well as eliminates pathogens and tumor cells[34].PGs are known as one of the inflammatory mediators and involved in diverse functions, including nerve growth,wound healing,and the immune response[3]. Therefore,the results that the sulfated SPS-CF is able to activate mac-ophages to produce pro-inflammatory mediators,TNF-α,IL-6,NO, and PGE2,suggest its potential use as an immunomodulatory agent. Taken together with the previously reported anti-gastric cancer activity[2],the immunomodulatory activity of the SPS-CF purified in this study demonstrates the pharmacological potential of the green alga,C.fulvescens.

In conclusion,SPS-CF is an acidic polysaccharide,a sulfated glucuronogalactomannan,with no proteinous materials.The results from the present study demonstrated that the polysaccharide SPS-CF purified from the Korean C.fulvescens,can modulate the immune function of RAW2.7murine macrophages and therefore it may be used as immune stimulators.

Acknowledgements

This study was supported by a grant from the Gyeonggi-do GRRC program and partly by the Research Fund,2009of The Catholic University of Korea.

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