Characterization of a natural hemagglutinin in the serum of a mud crab Scylla serrata ISJ 7: 79-88, 2010 ISSN 1824-307X RESEARCH REPORT Physico-chemical characterization of bacterial and hemagglutinins from the serum of the mud crab Scylla serrata SS Jayaraj1, R Thiagarajan2, M Arumugam2, S Vincent1 1Unit of Environmental Health and Biotechnology, Department of Advanced Zoology and Biotechnology, Loyola College, Chennai 600 034, India 2Unit of Pathobiology, Department of Zoology, University of Madras, Guindy Campus, Chennai 600 025, India Accepted February 15, 2010 Abstract A naturally occurring hemagglutinin (HA), with activity against bacteria and yeast cells were detected in the serum of Scylla serrata using mammalian erythrocytes (RBC), various bacteria and yeast as indicator cells. The serum gave highest HA titer with rabbit RBC, tripsinized yeast and Vibrio fluvialis. An analysis of the physico-chemical properties of the HA showed it to be specifically dependent on the presence of Ca2+ for its activity, stable between pH 7 to 9 and showed thermal stability between 10 to 30 °C. Further studies demonstrated that the HA is precipitable by ammonium sulphate and TCA. HA- inhibition assays performed with carbohydrates revealed that the serum HA was specific for non-reducing terminal glucose with 1-2 glucosidic linkages. Thus this agglutinin appears to be unique among all the known crustacean agglutinins. Key Words: hemagglutinin; bacterial agglutinin; yeast agglutinin; serum; mud crab; Scylla serrata Introduction Mud crab Scylla serrata is an economically important decapod crustacean in aquaculture in India. Understanding the defence system of S. serrata at humoral and cellular levels is indispensable for preventing disease occurrence. Lectins are carbohydrate-binding proteins and in invertebrates, lectins are vital means for non-self recognition and clearance of invading microorganisms. In invertebrates, phagocytosis is considered to be the primary mechanism of innate defense against foreign invaders (Ratcliffe and Rowley, 1981; Coombe et al., 1984; Ratcliffe et al., 1985). In this process, an intimate interaction of humoral substances, particularly as recognition factors, has been implicated (Richards and Renwrantz, 1991; Zelck and Becker, 1992). A variety of humoral factors, naturally occurring and/or formed after antigenic stimulation, have been detected in the serum of invertebrates and they include agglutinins (Cornick and Stewart, 1973; Renwrantz, 1986; Nalini et al., 1994; Murali et al., 1999; Jayasree, 2001; Jayaraj et al., 2008a), lysins (Osada et al., 1993), antibacterial (Xylander and Neverman, 1990), and antifungal proteins (Iijima et al., 1993), phenoloxidase system (Söderhäll, 1982), LPS binding protein (Jomori and Natori, 1992) and β- ___________________________________________________________________________ Corresponding author: SS Jayaraj Department of Advanced Zoology and Biotechnology Loyola College, Chennai, India E-mail: jayarajss@gmail.com 1, 3 glucan binding protein (Jayaraj et al., 2008b). Due to the probable functional similarities between agglutinins and vertebrate antibodies and the indications that agglutinins serve a defensive function (Ofek and Sharon, 1988), invertebrate agglutinins have been extensively studied. Agglutinins (= lectins) are di- or multivalent carbohydrate-binding proteins with the ability to agglutinate cells with complementary carbohydrates on their surfaces (Sharon and Lis, 1972; Barondes, 1988). They are known to specifically recognize the whole sugar (Bretting and Kabat, 1976), a specific site in a sugar (Shimizu et al., 1977), a sequence of sugars (Kobiler and Mirelman, 1980), or their glycosidic linkages (Koch et al., 1982). The agglutinating molecules are widely distributed in microorganisms (Sasmal et al., 1992), plants and animals (Gold and Balding, 1975). The body fluid or hemolymph of almost all invertebrate species tested contains agglutinins (Yeaton, 1981; Ratcliffe et al., 1985; Renwrantz, 1986; Nalini et al., 1994; Murali et al., 1999; Jayasree, 2001; Jayaraj et al., 2008a). The presence of agglutinins has also been detected in the mucus as well as in certain tissues of invertebrates (Renwrantz, 1986; Mullainadhan and Renwrantz, 1989; Suzuki and Mori, 1991). However, its immunological role is best understood in the hemolymph, and recent studies have shown that purified, hemolymph-derived agglutinins served as opsonin in a few insects and molluscs (Renwrantz and Stahmer, 1983; Pendland 79 mailto:jayarajss@gmail et al., 1988; Fryer et al., 1989; Richards and Renwrantz, 1991; Jayaraj et al., unpublished observations). Although a number of studies have demonstrated the presence of humoral agglutinins in several crustacean species, it can be noted that the immunological roles of these agglutinins remain largely unknown and that the carbohydrate specificity of serum agglutinins from crustaceans have been elucidated only in a few species (Vasta et al., 1983; Smith and Chisholm, 1992; Nalini et al., 1994; Kondo et al., 1998; Jayasree, 2001). This study thus describes RBC and bacterial binding activities, physico-chemical properties and carbohydrate specificity of a naturally occurring agglutinin in the serum of the marine crab S. serrata. Material and Methods Experimental animals and laboratory maintenance The marine crab Scylla serrata weighing 150 to 200 g were obtained from Muttukadu estuary, Chennai. In the laboratory, these crabs were maintained in plastic tanks (90 x 45 x 60 cm) containing aerated natural seawater and the medium was changed every day. The crabs were fed with Donax spp. during the period of acclimation (24 h) and only male crabs were used. Preparation of serum Hemolymph samples (1 to 2 ml) from individual crabs were collected from the cut end of the dactylus region of the walking leg. The samples were collected in clean polystyrene plastic tubes held on ice and allowed to clot at room temperature (RT: 28 ± 2 °C for 20 min). Serum was separated by centrifugation (400xg, 10 min, RT) and the resulting clear supernatant (=serum) was used immediately. Preparation of erythrocyte (RBC) suspension Human and other mammalian blood samples were obtained by venous or cardiac puncture and collected in sterile Alsever’s solution (Garvey et al., 1979) containing 10 µg/ml of streptomycin. Prior to use, the RBCs were washed thrice with 0.9% saline and once with TBS-I (50 mM tris-HCl, 115 mM NaCl, 10 mM CaCl2, 300 mOsm) by centrifugation (400xg, 5 min, RT). Unless specified, the RBC pellet was finally resuspended in TBS-I as 1.5% suspension (v/v). Preparation of yeast cell suspension A hundred mg commercial grade baker’s yeast (Saccharomyces cerevisiae) purchased from local market were suspended in 10 ml of 0.9 % saline, washed extensively with saline by centrifugation (400xg, 5 min, RT) and suspended in the same medium. The yeast cell suspension was heat- inactivated by autoclaving the suspension for 15 min at 15 psi. After cooling the suspension to RT, the heat-inactivated yeast cells were washed extensively with 0.9 % saline and finally resuspended in TBS-I as 0.5 % (v/v) suspension. Trypsinization of yeast cells Five μl of washed yeast cells were suspended in 1 ml of TBS-I containing trypsin (0.5 %) to give a final concentration of 0.5 % yeast. This suspension was incubated for 1 h at 37 oC with occasional gentle shaking. After incubation, the trypsinized yeast cells were washed once with TBS-I by centrifugation (400xg, 5 min, RT) and finally resuspended in TBS-I as 0.5 % (v/v) suspension. Hemagglutination (HA) assay HA assays were performed in V-bottom microtiter plates (Greiner, Nürtingen, Germany) by serial two-fold dilution of a 25 µl serum sample with an equal volume of TBS-I. After dilution, 25 µl RBC suspension was added to each well and incubated for 45 min at RT. The HA titers were recorded as the reciprocal of the highest dilution of the sample causing complete agglutination of RBC (Garvey et al., 1979). Controls for all assays consisted of the substitution of the sample by TBS-I. All the HA assays were performed in duplicate. Yeast agglutination assay The agglutinating activity of serum against yeast cells was performed in V-bottom microtiter plates by serial two-fold dilution of 25 µl serum with an equal volume of TBS-I. After dilution, 25 µl of 0.5 % native or trypsinized yeast cell suspension was added to each well and incubated for 45 min at 26 °C. Control consisted of substitution of serum with TBS-I. The agglutination of yeast cells by serum was assessed under microscope (40 x) and the agglutination titers were recorded as the reciprocal of the highest dilution of the sample causing complete agglutination. The experiment was performed using duplicates. Bacterial agglutinating activity Frozen stock culture of bacteria were inoculated in TBS-I and incubated for 6 h. The broth cultures were then centrifuged (5,000xg, 10 min). The pellet was collected and washed 3 times by centrifugation with TBS-I. The final concentration was adjusted to 1x108 cells ml-1 in TBS-I before use. Two-fold serial dilutions of serum samples (in duplicates) were made in TBS-I. Then, 25 µl of each serum dilution was incubated with 25 µl bacterial suspension. The reaction mixture was incubated at 20 ± 2 °C for 1 h. The appearance of clumps of bacteria was then recorded by microscopic examination (40x). Agglutination titer was defined as the reciprocal of the last dilution giving evidence of agglutination after incubation. The negative controls comprised mixed equal volumes of bacterial suspension and TBS-I. Divalent cation dependency and EDTA sensitivity Serum samples (each 300 µl in duplicates) were dialysed (MW exclusion limit <10,000) extensively at 20 °C against divalent cation-free TBS-II (50 mM tris-HCl, 135 mM NaCl, 300 mOsm) to examine cation dependency or in TBS-III containing 50 mM EDTA (50 mM tris-HCl, 72 mM NaCl, 40 mM CaCl2, 300 mOsm) to test EDTA sensitivity of the agglutinating activity of serum. The samples dialysed against TBS-III were subsequently re-equilibrated by dialysis in TBS-II. All the resulting dialysates were centrifuged (400xg, 10 min, 20 °C). The supernatant was tested for hemagglutinating activity using rabbit RBC in the presence of TBS 80 that did or did not contain 10 mM CaCl2, MgCl2 (or) MnCl2. A serum sample (300 µl) concurrently dialysed against, TBS containing 10 mM CaCl2 (TBS-I) was also tested for the hemagglutinating activity against rabbit RBC in TBS-I. pH and thermal stability The stability of serum HA activity (in duplicates) at different pH was examined by dialyzing (24 h, 4 °C) 300 μl serum samples against the following buffers at pH ranging from 3 to 12 (Lillie, 1954; Pearse, 1968): 0.2 M acetate buffer (pH 3 to 6), 0.2 M tris-HCl buffer (pH 7 to 9) and 0.1 M glycine- NaOH buffer (pH 10 to 12). After dialysis, all the samples were finally re-equilibrated by dialysis against TBS-I and the HA titer was determined with rabbit RBC. In another experiment designed to study the thermal stability of HA, 300 μl serum samples were held for 30 min at temperatures ranging from 10 to 100 °C, centrifuged and tested for HA activity with rabbit RBC. Precipitation by ammonium sulphate and trichloro acetic acid Precipitation of HA activity from serum (in duplicates) was attempted using 25, 50 and 75 % ammonium sulphate [(NH4)2SO4] solution as well as 10 % trichloroacetic acid (TCA) as described previously (Millar, 1987). The HA activity was finally measured using rabbit RBC. HA-inhibition assays Several carbohydrates were tested for their ability to inhibit serum HA activity. They were dissolved in TBS-III (50 mM tris-HCl, 115 mM NaCl, 50 mM EDTA, 300 mOsm) and if necessary, the pH was adjusted to 7.5 using concentrated NaOH. Serum samples were diluted with TBS-IV (50 mM tris- HCl, 5 Mm NaCl, 30 mM CaCl2, 135 mOsm) to a HA titer of 4 against rabbit RBC. The inhibitor to be tested (25 μl) was serially diluted two-fold with an equal volume of diluted sample in microtiter plates and in- cubated for 1 h at RT. Rabbit RBC suspension (25 μl) was added to each well and kept for 3 h at RT. The minimal concentration of carbohydrate that completely inhibited HA activity was recorded. The experiments were performed in duplicates. Protein determination Total protein concentration was measured using bovine serum albumin (BSA) as a standard (Lowry et al., 1951). Result Serum HA profile The serum of mud crab Scylla serrata agglutinated a variety of mammalian RBC types. Among the various RBC types tested, the highest titer of 128 was obtained with rabbit erythrocytes. The serum did not discriminate human A, B and O RBC types and agglutinated them to the same degree. Sheep and goat RBC were agglutinated at relatively low titers (Table 1). However serum did not agglutinate ox RBC and the serum showed highest agglutinating activity against tripsinized yeast cells when compare to native yeast cells (Table 2). Table 1 Hemagglutinating (HA) activity of serum from the mud crab S. serrata against various mammalian erythrocyte (RBC) types RBC types tested HA titer* Rabbit 128 Mice 64 Rat 32 Human B 32 Buffalo 16 Human A 8 Human O 8 Horse 4 Goat 2 Sheep 2 Ox 0 * Based on 20 determinations for each RBC type Bacterial agglutination The serum strongly agglutinated Vibrio fluvialis (titer 8), weekly agglutinated Vibrio parahemolyticus, Vibrio mimicus, Escherichia coli, Pseudomonas spp. and Aerobacter aerogenes. The results of bacterial agglutination was assessed using a phase-contrast microscope (Table 3). Divalent cation dependency an EDTA sensitivity The serum tested in TBS containing 10 mM CaCl2 (TBS-I) gave a hemagglutianation titer of 128 against rabbit RBC. When the serum was dialysed against TBS- I and then tested in the absence of divalent cation, the agglutination titer reduced to 16. But, this serum sample recovered it’s HA activity only upon addition of Ca2+ to the reaction mixture. Further, substitution of Ca2+ with Mg2+ showed a considerable improvement in HA titer, while Mn2+ could not reverse the effect of EDTA treatment. The serum dialyzed against TBS-III containing 50 mM EDTA and tested in the absence of divalent cation, considerably lost its agglutinating activity against rabbit RBC (Table 4). Further the addition of Mg2+ or 81 Mn2+ to this sample could not restore the original HA activity and addition of Ca2+, rescued the activity to 64 (Table 4). pH and thermal stability The serum hemagglutinating activity of the marine crab S serrata was tested in the pH range of 3-12. As shown in Figure 1, the hemagglutinating activity against rabbit RBC was found to be relatively stable between pH 7 and 9 reduced at pH below or above this pH range and completely lost at pH 11 and 12. The activity of the serum against rabbit RBC was unaffected only up to 30 °C but it was reduced considerably at 40 °C and 50 °C and completely inactivated at 60 °C and above (Fig. 2). Precipitation of serum HA activity by ammonium sulphate and TCA Serum was incubated with ammonium sulphate (25, 50 and 75 %) for 3 h. The 50 % concentration of ammonium sulphate completely precipitated HA activity from the serum, whereas ammonium sulphate concentration at 25 and 75 %, moderately precipitated the serum HA activity. By contrast 10 % TCA failed to precipitate serum HA activity (Table 5). Carbohydrate binding specificity Among the 24 carbohydrates tested, as many as 15 carbohydrates were found to inhibit serum hemagglutinating activity at concentrations ranging from 50 to 100 mM. All the three acetylated hexosamines (GlcNAc, GalNAc and ManNAc), but not their hexoses and hexosamine counterparts, were inhibitory at 50 or 100 mM. But the few sialic acids examined in this study and 9 other carbohydrates were not inhibitory when tested up to concentrations from 20 to 200 Mm (Table 6). Among the six different polysaccharides tested (Table7), only mannan and laminarin inhibited the HA activity at 0.25 and 0.50 mg/ml, respectively. Among all the inhibitory carbohydrates, mannan was found to be most potent. Table 2 Agglutinating activity of S. serrata serum against native and trypsinized yeast cells Yeast cells tested HA titer* Native 16 Trypsinized 64 * Based on 20 determinations for native and trypsinized yeast cells Discussion The serum of the marine mud crab S. serrata was found to possess naturally occurring agglutinating activity which showed the highest reactivity with rabbit RBC among the RBC types tested. These results also suggest that the RBC types agglutinated by the serum of S. serrata probably share a common surface receptor but with a quantitative difference in its HA binding sites. The serum agglutinated a variety of bacteria including Gram + and Gram- types and the species of Vibrio tested are known to be the most frequent opportunistic pathogens of aquatic crustaceans (Equidius, 1987; Vargas-Albores et al., 1993) and the plasma showed highest agglutinating activity against trypsinized yeast cells (Jayaraj et al., 2008b). The ability of the serum of S. serrata to agglutinate bacteria, particularly the potential pathogens, implicates a possible involvement of the humoral agglutinins in host defense response. Table 3 Agglutinating activity of S. serrata serum against various bacterial species Bacterial species tested Bacterial agglutination* (O.D: 0.8) Vibrio fluvialis 16 Vibrio alginolyticus 8 Vibrio vulnificus 8 Vibrio anguillarum 4 Vibrio parahemolyticus 4 Vibrio mimicus 2 Escherichia coli 2 Pseudomonas sp 2 Bacillus subtilis 4 Aerobacter aerogenes 2 * The assay was repeated six times for each bacterial species with identical results using samples from different preparations 82 Table 4 Effect of divalent cations and EDTA on the hemagglutinating (HA) activity of serum of S. serrata Serum sample tested Cation (10 mM) in sample diluting and RBC suspension HA titer* Before dialysis CaCl2 128 After dialysis against divalent cation free TBS (TBS-II). None CaCl2 MgCl2 MnCl2 16 128 64 16 After dialysis against TBS+10 mM CaCl2 (TBS-I) CaCl2 128 After dialysis against TBS+50 mM EDTA (TBS-III) followed by dialysis against TBS- II None CaCl2 MgCl2 MnCl2 8 64 4 4 * Determination using rabbit RBC and the results based on six determinations The serum agglutinin was heat-labile and susceptible to pH extremes. Its pH stability was comparable to that found in one earlier report (Nalini et al., 1994) and the proteinaceous nature of agglutinin has been well demonstrated (Mckay and Jenkin, 1969; Acton et al., 1969; Miller et al., 1972). The serum lost most of it’s HA activity after dialysis against cation-free TBS and when tested in the absence of cations. However, the activity in this sample completely regained only upon addition of Ca2+ and the HA titer of serum did not change after dialysis against TBS containing Ca2+. These observations demonstrated that the serum agglutinin of S. serrata specifically requires Ca2+ for it’s HA activity. Furthermore, the activity was sensitive to EDTA treatment, since dialysis of serum against TBS containing EDTA resulted in a significant reduction in the HA activity. None of the cations tested could restore the HA activity, albeit Ca2+ moderately rescued the activity in these samples, thereby indicating that the HA of S. serrata appears to be irreversibly sensitive to EDTA which is in contrast with other crustacean agglutinins (Hall and Rowlands, 1974a; Ravindranath et al., 1985; Kamiya et al., 1987). Crustacean serum agglutinins were shown to be specific for fucose (Amirante and Basso, 1984), glucose (Umetsu et al., 1991), galactose (Kamiya et al., 1987; Umetsu et al., 1991), GalNAc (Amirante and Basso, 1984; Vargas-Albores et al., 1993), or sialic acids such as NeuAc (Hall and Rowlands, 1974b; Vasta et al., 1983; Vasta and Cohen, 1984; Cassels et al., 1986; Ratanapo et al., 1990), 4- and 9-0-acetyl NeuAc (Ravindranath et al., 1985), 9-0- acetyl NeuAc (Vazquez et al., 1993) and NeuGc (Mercy and Ravindranath, 1993). The hemagglutination-inhibition test performed in this study using different carbohydrates, encompassing several diverse, unrelated monosaccharides and their derivatives as well as di- and oligo-saccharides inhibited the serum agglutinating activity. Furthermore, all the three acetylated hexosamines tested consistently inhibited the HA activities of crab Table 5 Ammonium sulphate and TCA precipitation of haemagglutination (HA) activity against rabbit RBC from the serum of S. serrata Ammonium sulphate and TCA (%) Saturation HA titer* (Rabbit RBC) Untreated Sample 128 25 % 16 50 % 128 75 % 32 TCA 10 % 0 * Determination using rabbit RBC and the results based on six determinations 83 Table 6 Inhibition of agglutinating activity (titer = 4) of serum from the mud crab S. serrata by various carbohydrates Carbohydrates tested Maximum concentration tested (mM) Minimum inhibitory concentration (mM)* Monosaccharides Simple sugars D-mannose 200 100 L-sorbose 100 100 D-fucose 100 100 L-fucose 100 50 Deoxy sugars L-rhamnose 200 100 N-acetyl sugars N-acetyl-D-glucosamine (GlcNAc) 200 100 N-acetyl-D-galactosamine (GalNAc) 200 100 N-acetyl-D-mannosamine (ManNAc) 200 50 Disaccharides Trehalose (glcα1 → 1 glc) 200 50 Cellobiose (glcβ1 → 4 glc) 200 100 β-gentiobiose (glcβ1 → 6 glc) 200 50 Sucrose 200 100 Palatinose (glcα1 → 6 fruc) 200 50 Melibiose (galα1 → 6 glc) 200 100 Lactose (galβ1 → 4 glc) 200 100 The following carbohydrates also did not inhibit the agglutinating activity and unless otherwise stated, all carbohydrate was tested at concentrations upto 200 mM: D-glucose, D-galactose, β-allose, D-fructose, D- glucosamine (GlcN), D-galactosamine (GalN), mannosamine (ManN), maltose (glcα1 → 4 glc), turanose (glcα1 → 3 fruc). * The assay was repeated five times for each carbohydrate with identical results Table 7 Agglutination–inhibition of S. serrata serum (agglutination titer = 4) by polysaccharides against rabbit RBC Polysaccharides tested Structural linkages Maximum concentration tested (mg. ml-1) Minimum inhibitory concentration (mg. ml-1)* Mannan (α 1-6 homopolymer of mannose) 1 0.25 Laminarin (β 1-3 homopolymer of glucose) 1 0.50 Dextran T70 (α 1-6,3,2 homopolymer of glucose) 2 NI Dextran T500 (α 1-6,3,2 homopolymer of glucose) 2 NI Inulin (α 2-6 homopolymer of fructose) 5 NI Colominic acid (α 2-8 homopolymer of Neu5Ac) 5 NI * The assay was repeated three times for each polysaccharide with identical results using samples from different preparations NI: No inhibition 84 Fig. 1 pH stability of hemagglutinating activity of serum of S. serrata against rabbit RBC serum and this has been earlier demonstrated (Amirante and Basso, 1984; Vargas-Albores et al., 1993; Mercy and Ravindranath,1993). The serum HA activity of S. serrata was not inhibited by the amino sugar tested. But it was inhibited by the simple hexoses namely mannose, L-sorbose, D- fucose and L-fucose. This finding is supported by previous studies wherein simple hexoses were shown to inhibit agglutinating activity, including fucose (Amirante and Basso, 1984), glucose (Umetsu et al., 1991). The C-l position of these hexoses is essential for interaction with the agglutinin. The amino derivatives (GlcN, GalN and ManN) did not inhibit the HA activity. However, their N-acetyl derivatives (GlcNAc, GalNAc and ManNAc) were able to inhibit the serum HA activity. Recently, Alpuche et al. (2005) have shown the inhibition of purified shrimp lectin by N- acetylated sugars and this lends support to our study. The disaccharides D-maltose and turanose failed to inhibit the HA activity but all other disaccharides were inhibitory. Fig. 2 Thermal stability of hemagglutinating activity of serum of S. serrata against rabbit RBC 85 All these observations clearly demonstrate that the presence of acetyl group at C-2 position of hexosamines does not favour the interaction with agglutinin whereas this position with a free hydroxyl group or its substitution with amino group is essential for the interaction. HA inhibition tests employing polysaccharides indicated that only laminarin and mannan inhibited the serum agglutinating activity. This indicates that the agglutinin molecules in crab serum tend to exhibit affinity for extended structures particularly for polysaccharides with β-linked hexoses. This is significant and shows the ability of the serum agglutinating activity to recognize a wide range of carbohydrates that will potentially help the animal to recognize a variety of pathogens based on their surface molecules (Zhang et al., 2009). Thus, all the results obtained from the inhibitory effects of various carbohydrates and glycoproteins taken together clearly indicate that the agglutinins present in the serum of S. serrata interact with a wide range of carbohydrates including acetylated hexosamines, acetylated or non-acetylated sialic acids and several other carbohydrates, though their preference for a specific carbohydrate structure could not be ascertained. However, these findings in turn strongly suggest the natural occurrence of multiple agglutinins in the serum of this crab. The agglutinins in several crustaceans have been characterized (Cornick and Stewart, 1968; Mckay and Jenkin, 1969, 1970; Huang et al., 1981; Vasta et al., 1983; Ratanapo et al.,1990; Adams, 1991; Nalini et al., 1994; Jayasree, 2001). This agglutinin appears to be unique among all the known crustacean agglutinins. 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