CvL, a lectin from the marine sponge Cliona varians: Isolation,
characterization and its effects on pathogenic
bacteria and Leishmania promastigotes
Raniere M. Moura
a, Alexandre F.S. Queiroz
a, Jacy M.S.L.L. Fook
b, Anny S.F. Dias
c,
Norberto K.V. Monteiro
c, Jannisson K.C. Ribeiro
c, Gioconda E.D.D. Moura
c,
Leonardo L.P. Macedo
c, Elizeu A. Santos
c, Maurício P. Sales
c,⁎
a
Departamento de Biofísica e Farmacologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
bDepartamento de Morfologia, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
cDepartamento de Bioquímica, Centro de Biociências, Universidade Federal do Rio Grande do Norte, Campus Universitário, 59072-970, Natal, RN, Brazil
Received 16 May 2006; received in revised form 16 August 2006; accepted 21 August 2006 Available online 30 August 2006
Abstract
CvL, a lectin from the marine sponge Cliona varians was purified by acetone fractionation followed by Sepharose CL 4B affinity chromatography. CvL agglutinated papainized treated human erythrocytes with preference for type A erythrocytes. The lectin was strongly inhibited by monosaccharideD-galactose and disaccharide sucrose. CvL is a tetrameric glycoprotein of 28 kDa subunits linked by disulphide bridges with a molecular mass of 106 kDa by SDS-PAGE and 114 kDa by Sephacryl S300 gel filtration. The lectin was Ca2+dependent, stable up to 60 °C for 60 min, with optimum pH of 7.5. CvL displays a cytotoxic effect on gram positive bacteria, such as Bacillus subtilis and Staphylococcus aureus. However, CvL did not affect gram negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa. Leishmania chagasi promastigotes were agglutinated by CvL up to 28titer. These findings are indicative of the physiological defense roles of CvL and its possible use in the antibiosis of bacteria and protozoa pathogenic.
© 2006 Elsevier Inc. All rights reserved.
Keywords: Sponge; Cliona varians; Pathogenic bacteria; Leishmania; Invertebrate marine
1. Introduction
Marine invertebrates survive in environments rich in bacterial populations that may be as high as 106and 109/mL (Austin, 1988), many of which may be pathogenic. The immune defence of these animals is non-specific with responses against microbial organisms based on both cellular (phagocytosis, encapsulation, respiratory burst, etc.) and humoral (agglutinins, lysozomal enzymes, antimicrobial factors, etc.) activities (Chu, 1988; Canesi et al., 2002). In marine animals, humoral lectins have been shown to participate actively, much the same as opsonins, in the phagocytosis of non-self-cells and molecules (Bayne, 1990). Lectins are ubiquitous proteins in the hemolymph and
cells of many invertebrates (Renwrantz, 1986; Cooper et al., 1992) with the activity based in the carbohydrate-binding protein that produces the agglutination of many cells, such as erythrocytes, bacteria, and others through interaction with specific complementary ligands (Goldstein et al., 1980; Sharon, 1984; Nesser et al., 1986). Moreover, there is evidence sup-porting the fact that marine invertebrate lectins are also involved in other endogenous biological events such as biomineralization (Goldstein et al., 1980) and embryonic development (Nesser et al., 1986; Bayne, 1990). Interestingly, it has been theorized that some marine invertebrate lectins mediate the interaction between symbiont and host (Vasta, 1992). Despite their ubiquity, their functions in nature are not fully clear. They represent a heterogeneous group of oligomeric proteins that vary widely in size, structure, molecular organization, and constitution of their combining sites. Many lectins belong to distinct protein groups
⁎ Corresponding author. Tel.: +55 84 32153416; fax: +55 84 32119208. E-mail address:[email protected](M.P. Sales).
1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.08.028
with similar sequences and structural features. They were initially classified into two different groups, C- and S-type lectins, based particularly on the carbohydrate-recognition domain (CRD), along with overall domain organization and physical–chemical properties, such as divalent cation depen-dence and free thiol requirement (Drickamer, 1988). Calcium dependence was the main property associated with C-type lectins, whereas thiol dependence was the characteristic associated to the S-type group. Recently, as a consequence of three-dimensional structure studies and the availability of additional primary sequence data, a more complete classification has emerged. Therefore, C-type lectins have been further cha-racterized into distinct subgroups, and S-type lectins reclassified as galectins; but the existence of new categories was also established (Barondes et al., 1994; Drickamer, 1995; Vasta et al., 1997; Vasta et al., 1999).
In most of the publications concerning anti-pathogenic activity in invertebrates, either single body compartments alone, such as haemolymph and egg masses, or extracts of whole bodies have been tested for antibiosis activity. Haug et al. (2004) recently showed that marine invertebrates possess antipathogen factors in several different tissues. The aim of the present paper was to describe the purification and cha-racterization of a galactose-binding lectin from the body of the sponge Cliona varians with respect to its chemical analysis, sugar specificities, and anti-pathogenic activities, revealing a plausible biological role of the lectin and use as an antibiotic. 2. Material and methods
2.1. Material
Papain, bovine trypsin and neuraminidase were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Human erythrocyte types A, B and O was donated by the Blood Bank, Hemocentro, Natal, Brazil. Bacteria were obtained from the collection donated by Dr. Patricia Soto-Mayor Sommer of Departamento de Biologia Celular e Genética of the Universi-dade Federal do Rio Grande do Norte, Natal, Brazil. Leishmania chagasi promastigotes were donated by Dr. Paulo Andrade of Departamento de Genética of the Universidade Federal de Pernambuco, Recife, Brazil.
2.2. Animal collection
Adult specimens of the marine sponge C. varians were collected in the littoral of Buzios beach, Parnamirim, Brazil. Specimens were collected and transported in ice to the laboratory and stored at 20 °C until use.
2.3. Haemagglutinating activity
The haemagglutinating activity was assayed in microtiter V plates (Nunc Brand products, Denmark) according to a twofold serial diluting procedure (Debray et al., 1981). The erythrocytes used were native or treated with papain and/or trypsin according toBenevides et al. (1998). One haemagglutinating unit (HU)
was defined as the amount of lectin able to agglutinate and hence precipitate the erythrocytes in suspension after 30 min. To each well was added 25 μL of the twofold serially diluted lectin solutions and 25μL of treated and untreated human erythrocytes (2% v/v suspension) and allowed to incubate for 30 min at room temperature. The degree of haemagglutinating activity was examined. The controls were set up with saline and erythrocytes and 1 mg/mL of Con A solution and erythrocytes.
2.4. Purification of marine sponge C. varians lectin (CvL) Specimens were cut into small pieces using sharp scissors. The pieces were ground 5–6 times with a mixer machine and stirred vigorously and extracted (1:2, w/v) with 0.05 M Tris– HCl buffer pH 7.5, for 2 h at room temperature. After centrifugation for 30 min at 12,000×g at 4 °C, the supernatant (crude extract) was precipitated with acetone at 0.5 vol, 1.0 vol and 2.0 vol. These fractions (F1, F2and F3) were then
freezer-dried and submitted to assays. The F2fraction showed a higher
level of haemagglutinating activity to human papainized erythrocytes type A. This fraction was applied to a Sepharose CL 4B (5.5 × 3.5 cm) affinity chromatography, equilibrated with 50 mM Tris–HCl, 20 mM CaCl2buffer, pH 8.0. The retained
proteins were eluted with 50 mM Tris–HCl, 100 mM EDTA, pH 8.0, at a flow rate of 30 mL h− 1. The retained peak, denominated CvL, was pooled and subjected to further analysis. Protein content of all fractions was measured as described byBradford (1976)and chromatography was monitored at 280 nm. 2.5. Carbohydrate specificity
The sugar specificity of the lectin was determined in the plate assay as described above. All inhibitors to be tested were dissolved in 150 mM NaCl at an initial concentration of 200 mM for monosaccharides (D-galactose, D-glucose, D-fructose, D
-fucose,D-ribose,D-arabinose,D-xilose,D-N-acetyl-glucosamine
andD-glucuronic acid) and disaccharides (maltose, sucrose and
lactose). An equal volume of the lectin solution was added to 25μL of the twofold serially diluted inhibitor solutions and the plate was incubated for 1 h at room temperature. Twenty-five microliters of human erythrocytes (2% v/v suspension) was added to each well and allowed to incubate for 30 min at room temperature. The degree of haemagglutination was examined and the maximum dilution of the inhibitor solution showing inhibition was recorded. The controls were set up with saline and erythrocytes, sugar and erythrocytes, and lectin and erythro-cytes. Results were expressed as the minimal sugar or glycoprotein concentration required to inhibit haemagglutinat-ing doses of the lectin.
2.6. Effects of metal ions, temperature and pH on haemagglutinating activity
The effect of pH on the lectin activity was studied by haemagglutinating assay of the lectin after dialysis against buffers of different pH ranging from pH 2.5 to pH 10.5 containing 100 mM CaCl2for 3 h. 50 mM glycine–HCl buffer
(pH 2.5), 50 mM. Acetate buffer (pH 4.5), 50 mM phosphate buffer (pH 6.5), 50 mM Tris–HCl buffer (pH 8.5), and 50 mM glycine–NaOH buffer (pH 10.5) were used.
The effect of divalent metal ions on the haemagglutinating activity of the lectin was assessed as follows. The lectin was dialyzed exhaustively against 50 mM EDTA followed by dialysis against 50 mM Tris–HCl buffer, pH 8.0. The haema-gglutinating activity was tested in the presence and in the absence of 100 mM Ca2+, Mg2+and Mn2+.
To study the effects of temperature on haemagglutinating activity, lectin (0.15 mg/mL) was serially diluted, incubated at −10, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 °C for 15, 30 and 60 min at each temperature and assayed after the haemagglutinating activity.
2.7. Sub-units determination, molecular mass estimation and staining glycoproteins
SDS polyacrylamide (7.5% and 12%) gel electrophoresis (SDS-PAGE) in the absence and presence ofβ-mercaptoethanol (0.1 M) was conducted as described by Laemmli (1970) to estimate the molecular mass of lectin and its sub-units by comparing of mobility of bands with protein molecular weight markers (kDa):β-galactosidase (116.0), BSA (66.2), ovalbumin (45.0), Lactate dehydrogenase (35.0), Restriction enzyme Bsp98I (25.0), β-lactoglobulin (18.4) and Lysozyme (14.4). Proteins were detected by staining with 0.1% Coomassie brilliant blue R-250 followed by silver staining according to
Blum et al. (1987), with modifications. Also, the molecular mass of CvL was estimated by Sephacryl S-200 gel filtration column (75 × 1.4 cm) calibrated with protein markers (kDa): potato β-amylase (Mr 150), bovine serum albumin (Mr 67), Hemoglobin (Mr 55) and trypsin inhibitor (Mr 21.5).
Staining glycoproteins in SDS-Polyacrylamide Gels was described byZacharius et al. (1969).
2.8. Antibacterial activity test
Four strains of pathogenic bacteria (Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa and Staphylococcus aureus) were selected for CvL agglutination. Bacteria were grown in LB broth (10 g bacto-tryptone, 5 g bacto-yeast extract, 10 g NaCl, 1 L H2O, pH 7·0). Cells in the exponential phase (15 h) were collected
and washed three times with 0.15 M NaCl solution by centrifugation at 6000×g for 10 min. The antibacterial activities were determined by continuous monitoring of bacterial growth. The test was performed in 1 mL plastic cuvette (Labcraft), in which 25μg mL− 1, 50μg mL− 1and 100μg mL− 1of CvL were incubated with 100μL of a suspension of bacteria culture diluted to a starting concentration of 5 × 103cells per cuvette. The growth chamber was maintained at 37 °C during the incubation period. The absorbance was measured at 1 h intervals for 4 h by a turbidometric method. Controls were added to assays: (1) bacteria plus 200μL of 150 mM NaCl solution; (2) bacteria plus 25 μg mL− 1, 50μg mL− 1and 100μg mL− 1of BSA; (3) bacteria plus 25μg mL− 1, 50μg mL− 1and 100μg mL− 1of CvL pre-incubated in 200 mM galactose solution.
2.9. L. chagasi promastigotes agglutination test
L. chagasi prosmatigotes were briefly maintained in modified Dwyer medium (Dwyer, 1972), with beef extract
Fig. 1. (A) Elution profile on Sepharose CL 4B of CvL. Approximately 1.6 mg of F1.0 was applied in the column (5.5 × 3.5 cm) equilibrated with 50 mM Tris– HCl buffer, containing 20 mM CaCl2, pH 7.5. The lectin was eluted with 50 mM
Tris–HCl buffer, containing 20 mM EDTA pH 7.5 and the fractions (1.5 mL each) and fractions were monitored at 280 nm. The haemagglutinating activity was detected in the presence of 2% human erythrocytes type A. (B) SDS-PAGE analysis of the (M) Molecular Weight Markers (kDa):β-galactosidase (116.0), BSA (66.2), ovalbumin (45.0), Lactate dehydrogenase (35.0), Restriction enzyme Bsp98I (25.0), β-lactoglobulin (18.4) and Lysozyme (14.4); CvL; βCvL in presence of β-mercaptoethanol (0.1 M). Proteins were stained with Coomassie blue and after with silver.
Table 1
Purification process of CvL by affinity chromatography on Sepharose 4B Fraction Total protein
(mg) Total HU Specific activity (HU/mg) Purification (x) Recovery (%) Crude extract 135 128 0.95 1.0 100 F2.0 19 256 13.5 14.2 14 CvL 1.4 256 182.9 192.5 1 One haemagglutinating unit (HU) was defined as the amount of lectin able to agglutinate and hence precipitate the erythrocytes in suspension after 30 min.
substitution. Defibrinated rabbit blood, yeast extract (5.0 g/l), and tryptone (0.1 g/l) were added to the basic medium. Mass production of the parasite was obtained by inoculating 1 l of RPMI 1640 to HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid) and 20 mL of rich promastigote mainte-nance culture were added to the fetal bovine serum (FBS). After harvest, the promastigotes were washed several times with cold Locke solution, treated at 37 °C with 0.4% trypsin for 45 min, and fixed for 20 h at 4 °C with 2% (w/v) formaldehyde in Locke solution. After being washed in saline, the parasites were stained with 0.1% Coomassie brilliant blue for 90 min. The parasite suspension was filtered through nylon gauze, and the concentration was adjusted to 5 × 107 cells/mL in 0.4% (w/v) formaldehyde. The agglutinating activity was assayed in microtiter V plates (Nunc Brand products, Denmark) according to a twofold serial diluting procedure. To each well were added 25 μL (1 mg mL− 1) of the twofold serially diluted lectin solutions and 25 μL of blue promastigotes and allowed to incubate for 24 h at 5 °C. The degree of agglutination was examined. Two controls were added: (1) saline and blue promastigotes; (2) CvL plus 200 mMD-galactose solution and blue promastigotes.
3. Results
3.1. Lectin isolation and CvL purification
Crude soluble protein extract obtained from marine sponge was initially precipitated at 0.5 vol, 1.0 vol and 2.0 vol with acetone and three protein fractions (F1, F2 and F3) were
obtained. The F2 protein fraction obtained showed strong
haemagglutining activity to papainized type A erythrocytes followed by papainized type B erythrocytes, while the other fractions exhibited low activity. The haemagglutining activity
of F2was activated by ions Ca2+. The F2was then applied to a
Sepharose affinity column and the retained peak was submitted to haemagglutining test (Fig. 1A). This purification procedure for marine sponge C. varians was observed by SDS-PAGE (Fig. 1B) and resulted in a high purification of 192.5 fold with a 1% yield (Table 1).
3.2. Physical and chemical analysis of the CvL
Electrophoretic analysis of CvL in the absence of a reducing agent (β-mercaptoethanol), showed one protein band with molecular mass of approximately 106 kDa, which after treatment withβ-mercaptoethanol decomposed to four subunits of 28 kDa (Fig. 1B). The glycoprotein staining procedure showed that CvL was marked as a magenta banding, a pattern characteristic of a glycoprotein (data not showed).
Fig. 2. Determination of molecular weight of CvL by gel filtration on Sephacryl S-300 chromatography. Proteins were applied in the column (127 × 2.6 cm) equilibrated with 50 mM Tris–HCl buffer, containing 20 mM CaCl2, pH 7.5.
The eluted protein fractions (1.5 mL each) were monitored at 280 nm. Protein molecular weight standards were: (a) potatoβ-amylase (Mr 150), (b) bovine serum albumin (Mr 67), (c) Hemoglobin (Mr 55) and (d) trypsin inhibitor (Mr 21.5).
Fig. 3. General properties of the CvL. The effects of (A) temperature, (B) pH and (C) Ca2+ on the haemagglutination activity of CvL.
The relative molecular mass of the native lectin was estimated at 114 kDa by gel filtration on a calibrated Sephacryl S-200 column (Fig. 2).
The study of the temperature effect on CvL showed that the lectin was stable at 60 °C (Fig. 3A). Preincubation of the
lectin in 6.0–8.0 pH range did not affect haemagglutinating activity (Fig. 3B).
Haemagglutinating activity of CvL was found to be Ca2+ dependent and an inhibition by 50 mM EDTA solution was reverted at 20 mM of CaCl2prepared in 150 mM NaCl solution (Fig. 3C).
3.3. Lectin specificity
Haemagglutinating activity of the CvL towards papainized Type A erythrocytes was mostly inhibited by D-galactose
(Table 2).
Fig. 4. Growth curves of B. subtilis (A) and S aureus (B) in the presence of the Cliona varians lectin (CvL). (○) control, bacteria plus 200 μL 0.15 M NaCl solution; (▲) control bacteria plus with BSA (100 μg/mL); (♦) bacteria plus CvL (25μg/mL), (
▪
) CvL (50μg/mL) and (•
) CvL (100μg/mL). Bacterial growth was measured from absorbance at 600 nm. Each point represents the mean of 3 estimates.Table 2
Inhibitory effect of monosaccharides, disaccharides and glycoprotein on haemagglutinating activity of CvL
Inhibitor compound MIC (mM) Monosaccharides D-Galactose 6.25 D-Glucose 25 D-Fructose NI L-Fucose NI D-Ribose NI D-Glucuronic acid 200 D-Arabinose NI D-Xilose NI D-N-acetyl-glucosamine NI Disaccharides Maltose 25 Sucrose 12.5 Lactose 12.5
MIC, minimum inhibitory concentration; NI, sugar not inhibitory until a concentration of 200 mM.
Fig. 5. Agglutination test for Leishmania chagasi promastigotes stained with 0.1% Coomassie brilliant blue. (A) Control, promastigotes plus 25μl 0.15 M NaCl solution; (B) promastigotes plus 25μg CvL and 25 μL 0.2 MD-galactose
3.4. Antibacterial and anti-Leishmania effects
The CvL Antibacterial activity was observed against gram positive bacteria, such as B. subtilis and S. aureus. The results showed that CvL, at a concentration of 25 μg/mL, exhibits high antibacterial activity (75%) on B. subtilis, which increases with higher concentrations; reaching 90% of inhibition with 100μg/mL, at 4 h (Fig. 4A). High antibacterial activity was also observed when CvL was incubated with S. aureus, reducing bacteria growth by 90% at 50μg/mL, at 4 h (Fig. 4B). The inhi-bitory effect of the lectin on the gram negative bacteria E. coli and P. aeruginosa was comparatively low. The agglutination test for L. chagasi promastigotes stained with 0.1% Coomassie brilliant blue showed that CvL was capable, according to a twofold serial diluting procedure, agglutinated up to 8 AU (agglutinating unit) titer, corresponding to the agglutination of 106cells by 1μg of CvL. The binding of CvL and promastigotes was not observed when the mixture was incubated in presence of D-galactose (Fig. 5A, B and C).
4. Discussion
Bioactive carbohydrates, proteins, peptides, and secondary metabolites have been isolated from various marine invertebrate classes, including the bioprospection of Porifera, which may be of tremendous potential benefit for humans. Among these bioactive molecules, lectins are proteins involved in the non-immune responses in invertebrate marines and their effects on the antibiosis of pathogenic organisms have been reported. In this study, a lectin with molecular mass around 114 kDa, formed by four subunits of 28 kDa linked by disulphide bridges, was isolated and purified to homogeneity from the sponge marine C. varian. The lectin, which was denominated CvL, exhibited A erythrocyte papainized specificity. CvL is a glycoprotein stable at pH range 6 to 8 and up to a temperature of 50 °C. The activity of this lectin was also found to be Ca2+dependent, as evidenced by complete activity loss in the presence of EDTA and the addition of Ca2+ ions regains the activity of the CvL. The presence of glycosylation and disulphide bridges in the protein was also indicative that CvL should be included in the C type lectin family, such as Cinachyrella alloclada (Atta et al., 1989), Pellina semitubulosa (Engel et al., 1992), Axinella polypoide (Buck et al., 1992) and Haliclona cratera (Pajic et al., 2002).
The binding property of the CvL was found to be preferential for galactose. A considerable number of lectins, including those isolated from sponges, were reported to react withD-galactose (Bretting et al., 1981; Schröder et al., 1990). Lectins with affinity for galactose appear to have important roles in modulating immune responses in marine animals (Yousif et al., 1994; Mistry et al., 2001; Kurata and Hatai, 2002). It has also been reported that several lectins isolated from various marine invertebrates can inhibit the growth of various bacterial pathogens and fungi (Tateno et al., 2002; Tasumi et al., 2004). CvL exhibited a strong anti-bacterial effect on pathogenic gram positive bacteria, such as B. subtilis and S. aureus whereas no effects were observed on pathogenic gram negative bacteria. This pattern of differentia-tion was observed among lectins from diverse origins because
almost all microorganisms express surface-exposed carbohy-drates as a potential lectin-reactive specific site. This property is exemplified in the invertebrate marine Tachypleus tridentatus, where several lectins, denominated tachylectins 1, 2, 3 and 4, were purified from their hemolymph. Tachylectin 1 was able to inhibit the growth of gram-negative bacteria (Saito et al., 1995). Tachylectin 2 had a binding specificity for N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) and promoted the agglutination of certain strains of gram-positive Staphylo-coccus (Okino et al., 1995; Kawabata and Iwanaga, 1999); Tachylectin 3 and Tachylectin 4 recognized the LPS of several gram-negative bacteria (Saito et al., 1997; Inamori et al., 1999). The binding of CvL to galactose sites in bacterial surface could explain deterrent effects found in agglutinating tests with gram-positive bacteria. The property of these lectins to select and complex with microbial glycoconjugates has made it possible to employ the proteins as probes and sorbents for whole cells, mutants and numerous cellular constituents and metabolites.
The few past years have witnessed an increasing interest in lectin–parasite interactions. This is because lectins have become valuable tools for studying the insertion, fate, distribution and function of glycoconjugates on and in parasites; for example, lectins are useful in defining various developmen-tal parasite cycles. This experiment showed that CvL had agglutinating activity on promastigotes of L. chagasi. This effect revealed that galactose receptors for lectin binding are present in this parasite stage. Parasite lectin specificity has been extensively studied in Trypanosomatids with respect to their lectin receptors (Jacobson, 1994; Gazzinelli et al., 1991; Schottelius and Alsien, 1994), for example, lectin 1B 4 of Griffonia simplicifolia, has been utilized to define terminal α-galactosyl-bearing glycoconjugates on T. cruzi, Leishmania braziliensis and Leishmania mexicana parasites (Petri et al., 1989; Souto-Padron et al., 1994). This is important because of the presence of anti-galactose antibodies that are found in increased amounts in the sera of patients infected with these parasites.
These findings showed that the isolation and characterization of proteins from marine animals has turned into a fast growing field in the life sciences because many marine bioactive molecules are attracting more and more attention due to their extensive physiological, biological and pharmacological use. The importance of marine biotechnology has grown immensely because of the achievements of these natural marine products. Acknowledgments
This work was supported by Brazilian Agencies: CAPES and CNPq. The authors thank Dr. Rosana Lucena de Sá Leitão, Dr. Dulce H. Seabra de Souza Silva and Silene Telma Lima de Santana, pharmacists from Hemonorte, Natal, RN, Brazil, for the generous blood bag donation.
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