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Keywords:

  • plasminogen;
  • Staphylococcus aureus;
  • triosephosphate isomerase

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Triosephosphate isomerase (TPI; EC 5. 3. 1. 1) displayed on the cell surface of Staphylococcus aureus acts as an adhesion molecule that binds to the capsule of Cryptococcus neoformans, a fungal pathogen. This study investigated the function of TPI on the cell surface of S. aureus and its interactions with biological substances such as fibronectin, fibrinogen, plasminogen, and thrombin were investigated. Binding of TPI to plasminogen was demonstrated by both surface plasmon resonance analysis and Far-Western blotting. It is suggested that lysine residues contribute to this binding because the interaction was inhibited by ɛ-aminocaproic acid. Activation of plasminogen to plasmin by staphylokinase or tissue plasminogen activator decreased in the presence of TPI, whereas TPI was degraded by plasmin. In other experiments, intact S. aureus cells had the ability to both increase and decrease plasminogen activation depending on the number of cells. Several molecules expressed on the surface of S. aureus were predicted to interact with plasminogen, resulting in its increased or decreased activation. These findings indicate that S. aureus sometimes localizes and sometimes disseminates in the host, depending on the molecules expressed under various conditions.

List of Abbreviations: 
C. neoformans

Cryptococcus neoformans

EACA

ɛ-aminocaproic acid

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GXM

glucuronoxylomannan

PVDF

polyvinylidene difluoride

SAK

staphylokinase

S. aureus

Staphylococcus aureus

S. pneumoniae

Streptococcus pneumoniae

SPR

surface plasmon resonance

t-PA

tissue plasminogen activator

TPI

triosephosphate isomerase

TSB

trypticase soy broth

Triosephosphate isomerase displayed on the cell surface of S. aureus acts as an adhesion molecule that binds to the capsule of C. neoformans, a fungal pathogen (1, 2). Previously, we have shown that TPI recognizes α-1,3-linked mannooligosaccharides of a size equal to or greater than that of triose. This structure is present in the backbone of GXM, which is the major component of the cryptococcal capsule.

Staphylococci, streptococci, and enterococci target extracellular matrix components such as collagen, fibronectin, laminin, and fibrinogen for adherence to, and colonization of, host tissues. Microbial surface components recognizing adhesive matrix molecules have common structural properties: a C-terminal domain anchoring the cell wall and an N-terminal signal peptide (3). Furthermore, several anchorless proteins exposed on the bacterial cell surface may act as adhesion molecules. Housekeeping proteins such as GAPDH and enolase, which are glycolytic enzymes, act as anchorless proteins interacting with the extracellular matrix of the host (4–6). For example, GAPDH, which is found in Streptococcus species (7, 8), Escherichia coli (9), and Candida albicans (10), binds fibrinogen, fibronectin, plasminogen, transferrin, laminin, and cytoskeletal proteins. Enolase on S. aureus, Streptococcus species, and Mycobacterium fermentans binds to plasminogen and fibronectin (6, 11–17).

Several studies have investigated TPI, although these studies are fewer in number than those for GAPDH and enolase. TPI on Paracoccidioides brasiliensis interacts with laminin and fibronectin. Thus, TPI may be important in adherence to, and invasion of, host cells, indicating that TPI may be a virulence factor for the pathogen (18). TPI of Schistosoma mansoni, a causative agent of parasitic infection, is a protective antigen in mice, suggesting that it may have potential as a vaccine candidate (19).

The presence of TPI on the cell surface of S. aureus has been suggested by proteomics analysis (20) and the results of our previous studies (1, 21). The secretory pathways of the glycolytic protein to the cell surface are unknown. However, the TPI gene is located in the same operon as GAPDH and enolase. The amino acid residue lysine common is common to the C-terminal of GAPDH, enolase, and TPI. The contribution of the C-terminal lysine residue of enolase to plasminogen activity has been demonstrated (12). To elucidate the function of TPI on the cell surface of S. aureus during infection, we performed a binding assay for TPI with several bioreactive host proteins using SPR analysis, which has been widely used to determine interactions of molecules, including GAPDH and enolase (7, 12).

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Interaction between triosephosphate isomerase and human bioreactive proteins

To determine the interaction between TPI and fibrinogen, fibronectin, thrombin, and plasminogen, SPR analysis was performed using a Biacore 3000 (GE Healthcare, Milwaukee, WI, USA). TPI was extracted from S. aureus RN4220 (supplied by K. Sekimizu, University of Tokyo, Japan) using 3 M lithium chloride and further purified by salting out (50% ammonium sulfate); the hydrophobic interaction was followed by anion exchange column chromatography, as described previously (2). The purity of the TPI was confirmed by SDS-PAGE, followed by silver staining. TPI was diluted with 10 mM sodium acetate buffer (pH 3.48) to a concentration of about 10 μg/mL and immobilized on a standard sensor chip (CM 5) using an amine coupling kit according to the manufacturer's instructions. Fibrinogen (human; Calbiochem, San Diego, CA, USA), fibronectin (human; Calbiochem), thrombin (human; Calbiochem), and plasminogen (human; Enzyme Research Laboratories, South Bend, IN, USA) were diluted to 800, 400, 200, 100, and 50 nM with HBS-EP running buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 3 mM EDTA, and 0.005% surfactant P20) and injected into the flow cell. The flow rate was maintained at 10 μL/min for immobilization and 20 μL/min for analysis.

In addition, a dot binding assay was performed to confirm the interaction shown by SPR. The spots of plasminogen, fibrinogen, fibronectin, and thrombin (400 nM, 30 μL) on a PVDF membrane were incubated overnight with purified TPI (approximately 500 nM). Because TPI has been shown to bind cryptococcal GXM, the membrane was incubated with GXM, followed by anti-C. neoformans rabbit antibody, a conjugate of anti-rabbit IgG and alkaline phosphatase, and then the substrate (1).

Binding between triosephosphate isomerase and plasminogen

Far-Western blotting was performed to confirm the interaction between TPI and plasminogen (22). TPI at three different levels of purification (crude extract, after salting out and after phenyl Sepharose CL-4B chromatography) was separated by SDS-PAGE and blotted onto PVDF membranes. The membranes were treated with plasminogen (100 nM), alkaline phosphatase-conjugated anti-plasminogen antibody (goat polyclonal antibody to human plasminogen; EY Laboratories, San Mateo, CA, USA), and alkaline phosphatase chromogenic substrate (15 mL of Tris–HCl buffer at 100 mM [pH 9.5] containing 100 mM NaCl and 5 mM MgCl2; 100 μL of 0.017%[w/v] nitroblue tetrazolium; 0.23%[v/v]N,N-dimethyl formamide; and 50 μL of 5% 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt in N,N-dimethyl formamide).

Interaction between triosephosphate isomerase and plasmin

Experiments were performed to determine the interaction between TPI and plasmin. TPI was first used as the ligand and plasmin as the analyte; however, degradation of TPI by plasmin was observed. Therefore, thereafter was used TPI as the analyte and plasmin as the ligand. Plasmin (human; Sigma, St. Louis, MO, USA) was diluted with 10 mM sodium acetate buffer (pH 5.0) and immobilized on a sensor chip (CM 5) using an amine coupling kit.

Degradation of triosephosphate isomerase by plasmin

To confirm degradation of TPI by plasmin, TPI was incubated with plasmin for 3 hr at 37°C, followed by SDS-PAGE and silver staining.

Plasminogen activation by staphylokinase or tissue plasminogen activator

To examine the effects of TPI on plasminogen activation, plasminogen was activated to plasmin using SAK or t-PA. The reagents were diluted in 50 mM Tris–HCl buffer (pH 7.4). First, 40 μL of 500 nM plasminogen and 10 μL of TPI (about 7 μg/mL) were incubated for 30 min at 37°C on a microplate. Next, 10 μL of SAK (recombinant human SAK, 5 μg/mL; Biological Industries, Haemek, Israel) or 10 μL of t-PA (recombinant human; 10 μg/mL; Technoclone GmbH, Vienna, Austria) were added and incubated for 10 min at 37°C, followed by the addition of the chromogenic substrate S-2251 (40 μL of 0.5 mM solution; Chromogenix, Chapel Hill, NC, USA). The release of p-nitroaniline from the substrate by plasmin activity was monitored as the absorbance at 405 nm at 10 min intervals for 3 hr.

Effects of intact live Staphylococcus aureus on plasminogen activation

To examine the effects of intact live S. aureus, cells were added to the above plasminogen activation assay system. After S. aureus RN 4220 had been cultured at 37°C for 18 hr in trypticase soy broth (TSB; Becton Dickinson, Franklin Lakes, NJ, USA) with shaking, stationary phase cell suspensions (10 μL) at various concentrations (1010, 5 × 109, 109, 108, and 107/mL) were incubated with 40 μL of 500 nM plasminogen for 30 min at 37°C in a 96-well microplate. After incubation with SAK or t-PA for 10 min at 37°C, substrate S-2251 was added, and the absorbance at 405 nm recorded.

Determining enolase and triosephosphate isomerase activities using intact live Staphylococcus aureus

The enzymatic activities of enolase and TPI in S. aureus cultured at 37°C for 18 hr in TSB were determined. For the enolase assay, cells were washed with assay buffer (50 mM Tris–HCl buffer [pH 7.8] containing 1 mM MgCl2), and different numbers of cells were incubated with 1 mM of 2-phosphoglycerate (Sigma). After 30 min incubation at 25°C, the cells were removed by centrifugation at 9700 g for 5 min at 4°C. The absorbance of the supernatant was measured at 240 nm. Enolase activity was expressed as the increase in absorbance depending on phosphoenolpyruvic acid production. For the TPI assay, cells were washed with assay buffer (30 mM triethanolamine–HCl buffer [pH 7.8] containing 1 mM EDTA), and different numbers of cells were incubated with 1.5 mM dl-glyceraldehyde 3-phosphate (Sigma), 0.1 mg of NADH, and 10 μg of glycerol 3-phosphate dehydrogenase (Wako, Osaka, Japan) in a final volume of 1 mL. After 5 min incubation at 25°C, the cells were removed by centrifugation at 9700 g for 5 min at 4°C. The absorbance of the supernatant was measured at 340 nm. TPI activity was expressed as the decrease in absorbance due to NADH consumption.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Interaction between triosephosphate isomerase and bioreactive proteins

In the SPR analyses, fibrinogen, thrombin, and fibronectin showed weak responses for TPI, whereas plasminogen bound in a dose-dependent manner (Fig. 1). Kinetic analysis revealed two equilibrium constants, KD1= 3.18 × 10−10 M and KD2= 3.12 × 10−7 M, from the heterogeneous ligand-parallel reaction model using BIA evaluation software, since the model fit. Moreover, the interaction was inhibited by EACA used as a lysine analog at concentrations of 10 and 100 nm in a dose-dependent manner (Fig. 2), suggesting that lysine residues contribute to the binding. The responses were markedly weaker to fibrinogen, thrombin, and fibronectin than to plasminogen. Because different analytical methods were required to confirm the response, a dot binding assay was performed; the results supported the interactions observed in the SPR analyses. As shown in Fig. 3, plasminogen clearly binds with TPI. In addition, fibronectin had a slightly positive reaction, indicating that binding with fibronectin is not negligible.

image

Figure 1. Sensorgrams from the SPR analyses showing the interaction between TPI from S. aureus and fibrinogen, thrombin, fibronectin, or plasminogen. TPI from S. aureus was the ligand, and the analytes were (a) fibrinogen (50–800 nM), (b) thrombin (50–800 nM), (c) fibronectin (50–400 nM), and (d) plasminogen (50–800 nM).

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image

Figure 2. Effects of EACA on the interaction between TPI and plasminogen using SPR analyses. TPI from S. aureus was the ligand and the analyte was plasminogen (400 nM) with 0, 10, or 100 nM EACA.

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image

Figure 3. Binding of TPI and several human proteins in the dot assay. Immobilized proteins were incubated with TPI followed by cryptococcal GXM, anti-GXM rabbit antibody, conjugate of anti-rabbit antibody, alkaline phosphatase and substrate. The lower membrane was a control that was incubated without TPI. Blue spots indicate a positive reaction.

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Binding between triosephosphate isomerase and plasminogen

The interaction between TPI and plasminogen observed in the SPR analyses was examined by Far-Western blotting. At least two protein bands of about 27 kDa and 47 kDa that bound to plasminogen were observed in the crude extract (Fig. 4). The smaller band was considered TPI based on our previous experiments (2). The density of the 47 kDa band decreased during TPI purification.

image

Figure 4. Detection of plasminogen-binding proteins by Far-Western blotting analysis. Lane 1, TPI in crude extract; lane 2, TPI fraction after salting out with 50% ammonium sulfate; lane 3, TPI purified by phenyl Sepharose CL-4B chromatography.

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Interaction between triosephosphate isomerase and plasmin

With TPI as the immobilized ligand, the sensorgram response units decreased on injection of plasmin as the analyte, suggesting that the reaction is dependent on the proteolytic activity of plasmin. To confirm the interaction of TPI with plasminogen, we immobilized plasmin and injected TPI. As shown in Fig. 5a, the interaction of plasmin with TPI is dose-dependent, suggesting that plasmin binds to TPI.

image

Figure 5. Interaction between TPI and plasmin. (a) Sensorgrams from SPR analyses. Plasmin was the ligand and the analyte was TPI (22–215 nM). (b) SDS-PAGE, followed by silver staining. Lane 1, plasmin (4 ng); lane 2, partially purified TPI by phenyl Sepharose CL-4B chromatography; lane 3, partially purified TPI incubated with plasmin (4 ng) at 37°C for 3 hr before electrophoresis.

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Degradation of triosephosphate isomerase by plasmin

To verify the degradation of TPI by plasmin, we performed SDS-PAGE. In the presence of plasmin, the band corresponding to TPI disappeared (Fig. 5b, lane 3). TPI possesses 20 lysine residues within a sequence of 253 amino acids (positions 11, 14, 20, 33, 54, 57, 91, 113, 114, 120, 140, 150, 151, 162, 178, 202, 217, 222, 241, and 253). The abundance of lysine residues in TPI may lead to its proteolysis by plasmin, because lysine is the cleavage site for plasmin. Concerning the 14, and 21 to 23 kDa-protein bands, which also disappeared after plasmin treatment, we speculate that these represent TPI that had partially degraded during storage at 4°C, although no evidence for this is available.

Plasminogen activation by staphylokinase and tissue plasminogen activator

We examined the effects of TPI binding on activation of plasminogen to plasmin by SAK or t-PA. The absorbance indicating plasmin activity was significantly delayed in the presence of TPI compared with that in its absence (Fig. 6). Therefore, TPI may decrease plasminogen activation induced by both SAK and t-PA. In preliminary tests with smaller amounts of TPI, the activities were not affected significantly.

image

Figure 6. Effects of TPI on activation of plasminogen by SAK or t-PA. (a) Plasminogen was preincubated with TPI for 30 min at 37°C and then SAK was added. (b) Plasminogen was preincubated with TPI for 30 min at 37°C and then t-PA was added. After incubation for 10 min, substance S-2251 was added. The absorbance at 405 nm was monitored for 3 hr at 10 min intervals.

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Live intact S. aureus were used in the system instead of TPI (Fig. 7). In both the SAK and t-PA activation systems, plasminogen activation increased in the presence of many bacterial cells and decreased when relatively fewer cells were present. However, the cells did not activate plasminogen without SAK or t-PA.

image

Figure 7. Effects of staphylococcal cells on the activation of plasminogen by SAK or t-PA. Plasminogen was preincubated with various numbers of cells for 30 min at 37°C and then (a) SAK or (b) t-PA was added. After 10 min incubation, substance S-2251 was added. The absorbance at 405 nm was monitored at 10 min intervals.

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Enolase and triosephosphate isomerase activities using whole cells

Enolase and TPI enzymatic activities were tested using whole cells to detect enolase and TPI on the S. aureus surface. Figures 8a and 8b show enolase and TPI activities, respectively. Enolase and TPI activities were demonstrated as increasing and decreasing absorbance, respectively, and both occurred in a dose-dependent manner. To investigate whether enolase and TPI activities were localized to the cell surface, the cells were pretreated with formalin (final concentration 1%, 15 min), and the activities examined. The surface enzymes were assumed more sensitive to denaturation (23). Enolase and TPI activities were reduced to 52 and 26%, respectively (cells, 4 × 107), by this treatment, suggesting the involvement of surface enzymes.

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Figure 8. Detection of enolase and TPI enzymatic activities using intact staphylococcal cells. (a) Enolase activity is shown by an increase of absorbance at 240 nm. (b) TPI activity is indicated by a decrease in absorbance at 340 nm resulting from consumption of NADH.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

Proteins localized on the surfaces of microbes play important roles in interactions with other cells and molecules. In the process of infection, these proteins can be key substances for adherence to, and invasion of, the host (20, 24, 25). Glycolytic enzymes act in the cytoplasm of microbes, but accumulating evidence indicates that some glycolytic enzyme proteins, such as GAPDH, enolase, and TPI, are present on the cell surface, where they may have multiple functions (5, 6). Several of these enzymes interact with proteins in the plasma. For example, in Gram-positive bacteria, GAPDH of Streptococcus pneumoniae, Streptococcus pyogenes, and Streptococcus equisimilis is a plasminogen-binding protein (7, 26, 27), suggesting the possibility of immunogenic GAPDH as a candidate for a pneumococcal vaccine (8). In Gram-negative bacteria, GAPDH is thought to act as a virulence factor and may contribute to the pathogenesis of enterohemorrhagic E. coli and enteropathogenic E. coli by binding to human plasminogen, fibrinogen, or intestinal epithelial cells (9). In the fungus Candida albicans, GAPDH on the outer surface of the cell wall is able to adhere to fibronectin and laminin (28). Enolase from Streptococcus, Staphylococcus, and Lactobacillus species interacts with plasminogen and laminin (6, 29, 30).

Previously, we reported that TPI of S. aureus is a lectin and that it binds to the fungal pathogen C. neoformans via a capsular polysaccharide, GXM. The attachment of S. aureus to C. neoformans induces apoptosis-like cell death of the fungal pathogen (1, 31, 32). TPI recognizes at least three mannose residues in the α-1,3-linked mannan backbone of GXM. Furthermore, TPI may have two binding pockets for mannooligosaccharides (2). As TPI appeared to be a multifunctional protein, we investigated the interaction of TPI with proteins in the plasma.

Here, we have shown that TPI is a plasminogen-binding protein. Moreover, Far-Western blotting analysis revealed a plasminogen-binding protein in addition to TPI in the crude extract. Based on size, we speculate that the band at about 47 kDa is enolase. Enolase in S. aureus binds plasminogen and enhances its activation to plasmin (33). Recently, enolase was proposed as a candidate for a vaccine against S. aureus infection (34).

An interaction between TPI expressed on the cell surface of the fungus Paracoccidioides brasiliensis and laminin has been suggested (18). Our preliminary experiments indicated binding between staphylococcal TPI and laminin by SPR. However, further study would help to clarify the multiple functions of TPI.

In the kinetic analysis of the TPI and plasminogen interaction, two equilibrium constants, KD1 (3.18 × 10−10) and KD2 (3.12 × 10−7), were calculated from the heterogeneous ligand-parallel reaction model. Bergmann and Hammerschmidt reported the equilibrium constants of S. pneumoniae GAPDH for plasminogen (KD1= 4.3 × 10−7 M and KD2= 1.6 × 10−10 M) and for plasmin (KD1= 2.8 × 10−8 M and KD2= 5.2 × 10−8 M) (7). The binding affinities of S. pneumoniae enolase for plasminogen have been calculated as 8.6 × 10−8 M and 5.5 × 10−10 M (12). Thus, GAPDH, enolase, and TPI show multiple binding sites and similar affinities for plasminogen. The multiple binding sites could facilitate higher affinity fitting between the molecules. In our experiments the interaction of TPI and plasminogen was inhibited by EACA, a lysine analog. The common amino acid residue at the C-terminus of these enzymes is lysine. For enolase from S. pneumoniae, Bergmann et al. have shown that, in addition to the C-terminal lysyl residue being the binding site for the kringle motif of plasmin(ogen), nine amino acid residues (residues 248–256) form a second binding site (12). Recently, Cork et al. reported that surface enolase group A Streptococcus lysine residues at positions 252, 255, 434, and 435 play a concerted role in plasminogen acquisition (35).

We examined whether TPI could affect the activity of plasminogen. Enolase, GAPDH, and known plasminogen-binding proteins of S. pneumoniae promote invasion of pathogens into the host tissue via binding with plasminogen to initiate its proteolytic activity (6, 16, 36–38). However, in our experiments, TPI decreased the conversion of plasminogen to plasmin, suggesting involvement of TPI in the inhibition of fibrinolysis. In contrast, staphylococcal enolase activates plasminogen (6, 39, 40). Here, we also observed that cleavage of TPI by plasmin. S. aureus produces a large number of virulence factors. Some of these factors are toxins secreted into the extracellular environment, including exoenzymes, whereas others are components of the cells. The well-known coagulase and SAK seem to have opposite functions. The former plays a role in the formation of fibrin, whereas the latter acts in fibrinolysis. The plasminogen activation experiments using intact cells instead of purified TPI suggested that the overall phenomenon is affected by the number of cells. Plasminogen was activated in the presence of many bacterial cells, whereas it was delayed in the presence of fewer cells. The activities of enolase and TPI were determined to ascertain whether they are expressed on the cell surface. At similar concentrations of cells enolase and TPI were detected, although a quantitative investigation remains to be conducted. Glycolytic enzymes may be involved in the coordinated activities of coagulation and fibrinolysis. These proteins may serve multiple functions on the cell surface in addition to glycolysis (41), although the expression and posttranscriptional processing of each enzyme's actions on the cell wall are unknown.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

This work was supported by a grant from the High-Tech Research Center Project, the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (S0801043).

DISCLOSURE

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES

None of the authors have any conflicts of interest associated with this study.

REFERENCES

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  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. DISCLOSURE
  8. REFERENCES
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