From the Division of Biochemistry and Molecular
Biology, Faculty of Science, Australian National University, Canberra
ACT 0200, Australia, and the § University Laboratory of
Physiology, Parks Road, Oxford OX1 3PT, United Kingdom
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ABSTRACT |
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The growth of the human malaria parasite, Plasmodium falciparum, within its host erythrocyte is reliant on the uptake of a number of essential nutrients from the extracellular medium. One of these is pantothenic acid, a water-soluble vitamin that is a precursor of coenzyme A. In this study we show that normal uninfected erythrocytes are impermeable to pantothenate but that the vitamin is taken up rapidly into malaria-infected cells via a transport pathway that has the characteristics (furosemide sensitivity, nonsaturability) of previously characterized, broad specificity permeation pathways induced by the intracellular parasite in the host cell membrane. The transport of pantothenate therefore constitutes a critical physiological role for these pathways. Inside the parasitized cell pantothenate undergoes phosphorylation, the first step in its conversion to coenzyme A. Parasites within saponin-permeabilized erythrocytes were shown to take up and phosphorylate pantothenate, consistent with the intracellular parasite having both a pantothenate transporter and a pantothenate kinase. Comparisons of the rate of phosphorylation of pantothenate by lysates prepared from uninfected and infected erythrocytes revealed that the pantothenate kinase activity of the P. falciparum trophozoite is some 10-fold higher than that of its host cell and that most, if not all, of the phosphorylation of pantothenate within the malaria-infected cell occurs within the intracellular parasite. These results contrast with those of previous studies in which it was proposed that the avian malaria parasite Plasmodium lophurae lacks pantothenate kinase (as well as the other enzymes for the synthesis of coenzyme A) and is reliant upon the uptake of preformed coenzyme A from the host cell cytosol.
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INTRODUCTION |
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Malaria is caused by a protozoan parasite (genus Plasmodium) which, during the course of its life cycle, invades the red blood cells of its vertebrate host. This strategy serves to protect the parasite from attack by the immune system of the host; however, it poses significant challenges to the protozoan with regard to obtaining essential nutrients from the plasma.
One such compound is pantothenic acid, a water-soluble vitamin (Mr 219; Fig. 1) that serves as a precursor of the enzyme cofactor coenzyme A. Early evidence for the importance of exogenous pantothenate in supporting the growth of the intracellular malaria parasite came from the observation that in chickens infected with the avian parasite Plasmodium gallinaceum a dietary deficiency of pantothenate resulted in a marked reduction in the parasitemia (1). Forty years later it was demonstrated in in vitro growth assays that the sustained growth of the human malaria parasite Plasmodium falciparum within human erythrocytes is dependent upon there being pantothenate in the extracellular medium (2). The requirement of the intracellular malaria parasite for an exogenous supply of pantothenate implies that there is a mechanism by which this compound can gain entry into the infected erythrocyte. However, this process has not been characterized.
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In mammalian tissues, as well as in bacteria, pantothenate is converted to coenzyme A via a series of enzyme-mediated steps, the first of which involves the phosphorylation of pantothenate by pantothenate kinase (3, 4). In a series of studies with Plasmodium lophurae-infected duck erythrocytes Trager and colleages (5, 6) obtained evidence that all five enzymes involved in the conversion of pantothenic acid to coenzyme A are present in the erythrocyte cytosol of both parasitized and nonparasitized duck red cells but absent from the intracellular parasite. This led to the proposal that in malaria-infected erythrocytes pantothenic acid is converted to coenzyme A within the host cell cytosol, from which it is taken up by the intracellular parasite. Support for this model came from the earlier observation that the survival of erythrocytic stages of P. lophurae removed from their host red cells is favored by the inclusion of coenzyme A (but not by the inclusion of pantothenate) in the extracellular medium (7, 8). It remains to be established, however, whether the same model is applicable in the case of P. falciparum-infected human erythrocytes.
In this study we have investigated the transport and subsequent phosphorylation of pantothenate in human erythrocytes infected in vitro with P. falciparum. Our results indicate that the mature human erythrocyte lacks a functional pantothenate transporter but that the vitamin enters malaria-infected erythrocytes rapidly via the broad specificity, new permeation pathways (NPP)1 induced by the parasite in the infected cell membrane (9-13). Once inside the parasitized cell, pantothenate undergoes phosphorylation. Data from experiments carried out with saponin-permeabilized parasitized erythrocytes and with cell lysates and extracts indicate that the intraerythrocytic form of P. falciparum has the ability to take up and metabolize pantothenate and that most, if not all, of the phosphorylation of pantothenate within the parasitized erythrocyte occurs within the intracellular parasite. The model proposed previously for P. lophurae-infected avian erythrocytes in which the parasite is wholly reliant upon a supply of preformed coenzyme A from the host red cell cytosol is therefore not applicable in the case of P. falciparum-infected human erythrocytes.
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EXPERIMENTAL PROCEDURES |
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Cell Culture and Preparation--
Human erythrocytes (type O)
infected with either the ITO4 (14) or FAF6 (15) lines of P. falciparum were cultured under 1% O2, 3%
CO2, 96% N2 in RPMI 1640 culture medium (Life
Technologies, Paisley, UK or JRH Biosciences (CSL), Melbourne,
Australia) supplemented with HEPES (40 mM), glucose (10 mM), glutamine (2 mM), gentamicin sulfate (25 mg liter1), and either human serum (8.5% v/v, pooled
from different blood donors) or Albumax (0.5% w/v (16)). All
experiments with parasitized erythrocytes were carried out using
trophozoite-infected cells (approximately 35 h postinvasion),
synchronized using a combination of sorbitol hemolysis (17) and gelatin
flotation (18). For experiments with intact infected erythrocytes the
parasitized cells were concentrated immediately before experimentation
by layering 20 ml of infected cell suspension onto 20 ml of Percoll (diluted with 10 × phosphate-buffered saline and water to give a
density of 1.090 and an osmolality of 320 mosm (kg
H2O)
1) and centrifuged for 10 min at 400 × g. Cells harvested from the top of the Percoll layer
(having parasitemias usually in the range of 85-95%) were washed
immediately (four times for 5 min each, 600 × g) in
saline (150 mM NaCl, 5 mM KCl, 10 mM HEPES, and 5 mM glucose). In a number of
cases cells harvested from beneath the Percoll layer (having
parasitemias
5%) were used as a control to establish that the
phenomenon of interest was restricted to the parasitized cells in the
malaria-infected cell suspensions.
Saponin Permeabilization of Parasitized Erythrocytes-- In a number of experiments parasitized erythrocytes were permeabilized by treatment with saponin, a plant-derived glycoside, using a method modified from that described by Hsiao et al. (19) Saponin interacts with cholesterol in the erythrocyte membrane to form lytic pores, but it leaves the parasite plasma membrane (which lacks cholesterol) intact.
Saponin (0.05% w/v) was added to parasitized erythrocytes (hematocrit ~10%) suspended in RPMI supplemented with glucose (20 mM) and HEPES (25 mM) at 37 °C. The suspension was centrifuged immediately (2,060 × g, 10 min). The supernatant solution (containing hemoglobin and other red cell cytosol components, together with the plasma membranes of uninfected cells) was aspirated, and the resulting pellet was washed (three times for 10 min each, 2,060 × g, 50 ml) and then resuspended in an appropriate volume of RPMI prewarmed to 37 °C. Approximately 92% of the parasites within the permeabilized cells excluded trypan blue, consistent with their plasma membrane remaining intact.Uptake Measurements in Intact Erythrocytes--
Uptake of
pantothenate into intact infected and uninfected erythrocytes was
monitored using [14C]pantothenate (NEN Life Science
Products). All uptake measurements were carried out using cells washed
(three times) and resuspended in RPMI 1640 at 37 °C. Cell
suspensions were aliquoted into microcentrifuge tubes, and uptake
commenced with the addition to the cell suspension of
[14C]pantothenate (at 0.1 µCi ml1),
giving a final extracellular pantothenate concentration of 3 µM. The final cell count was, in most experiments,
approximately 2 × 108 cells ml
1. At
appropriate time intervals aliquots (typically 0.15 ml) of the
suspension were transferred to microcentrifuge tubes which, with the
exception of those experiments in which the degree of phosphorylation
of intracellular solute was determined (see next section), contained
ice-cold RPMI (typically 0.45 ml) supplemented with 0.2 mM
furosemide and layered over 0.2 ml of dibutyl phthalate. The tubes were
centrifuged immediately (10,000 × g, 50 s) to
sediment the cells below the oil, thereby terminating the uptake of
radiolabeled solute.
Monitoring the Phosphorylation of Pantothenic
Acid--
Phosphorylation of pantothenate by intact erythrocytes,
saponin-permeabilized cells, and cell lysates was estimated using a
combination of ZnSO4 and Ba(OH)2 (Somogyi
reagent) which acts to precipitate phosphorylated compounds from
solution (21, 22). In preliminary experiments it was shown that
treatment of a solution of [14C]pantothenate with Somogyi
reagent had no effect on the concentration of radiolabel in the
solution, whereas treatment of a solution of the phosphorylated
compound [35S]ATPS resulted in a 99.5% depletion of
radiolabel from the solution (not shown).
Phosphorylation of Pantothenate within Intact Parasitized Erythrocytes-- In experiments designed to monitor the phosphorylation of pantothenate taken up into intact cells it was necessary to ensure that all intracellular metabolism ceased at the time of sampling. In experiments of this sort with intact infected erythrocytes, uptake was halted by transferring aliquots of cell suspension to microcentrifuge tubes containing 0.5 ml of dibutyl phthalate layered over 0.5 ml of a 5.6% w/v solution of perchloric acid (PCA) made up in a saturated sucrose solution, then immediately centrifuging the tubes (10,000 × g, 50 s). The cells passed through the oil and into the PCA solution, thereby terminating the uptake, lysing the cells, and precipitating the cell protein. The tubes were washed (four times) as described above, then the dibutyl phthalate was removed. A 0.4-ml aliquot of the extract solution was transferred to a clean microcentrifuge tube where it was neutralized and then divided into two for quantitation of [14C]pantothenate phosphorylation using Somogyi reagent, as described above.
Uptake and Phosphorylation of Pantothenate by Saponin-permeabilized Erythrocytes-- Initial attempts to adapt the method described above (in which uptake and metabolism were halted instantaneously by centrifuging the cells through an oil layer into a dense PCA solution) for use with saponin-permeabilized erythrocytes were unsuccessful; the permeabilized cells have a lower density than intact cells and did not enter the PCA solution. In these experiments the metabolism of pantothenate was therefore halted by chilling the samples to 0 °C, at which temperature phosphorylation of pantothenate does not occur (see "Results").
Saponin-permeabilized infected cells suspended in RPMI (at 37 °C) were combined with an equal volume of RPMI containing 0.2 µCi/ml [14C]pantothenate, giving a final activity of 0.1 µCi/ml and a final extracellular pantothenate concentration of 3 µM. At appropriate time intervals 0.3-ml aliquots of the permeabilized cell suspension were transferred into microcentrifuge tubes containing an oil layer (a 5:4 blend of dibutyl phthalate:dioctyl phthalate, having a density of 1.015 g/ml). The tubes were centrifuged immediately (15,800 × g, 2 min) and then chilled to 0 °C in an ice slurry. The supernatant was aspirated and the radioactivity remaining on the walls of the tube removed by rinsing the tubes five times with water. The oil was aspirated and the pellet solubilized in 0.5 ml of 0.5% Triton X-100, then deproteinized by the addition of 5.6% w/v PCA (0.5 ml) followed by centrifugation (10,000 × g, 10 min). A 0.8 ml-aliquot of the supernatant was neutralized with 5% KOH then divided into two for quantitation of [14C]pantothenate phosphorylation using Somogyi reagent, as described above.Phosphorylation of Pantothenate by Cell Lysates and Extracts-- The presence of pantothenate kinase in the cytosol of normal uninfected erythrocytes and in the host cell and parasite compartments of trophozoite-infected cells was investigated using cell lysates and extracts prepared from: (i) high (>85%) parasitemia-infected cell suspensions; (ii) saponin-permeabilized parasitized erythrocytes; (iii) the erythrocyte cytosol fraction of saponin-permeabilized parasitized erythrocytes; (iv) a suspension of predominantly uninfected cells (3-5% parasitemia) harvested from infected cell suspensions (collected from beneath the Percoll layer used to generate suspensions having high parasitemias); (v) normal, uninfected erythrocytes, cultured in parallel with infected cell suspensions.
Phosphorylation of [14C]pantothenate by the various cell extracts was measured under conditions similar to those used by Bennett and Trager (5) in their original study of pantothenate metabolism by P. lophurae-infected duck erythrocytes. Cell lysates (from intact parasitized erythrocytes, saponin-permeabilized parasitized erythrocytes, uninfected erythrocytes) were prepared by diluting the cell suspension 1/10 in a 10 mM phosphate buffer, pH 7.4, followed by trituration (10 times) through a 25-gauge needle. The lysates were then centrifuged three times at 2,000 × g for 30 min, each time transferring the supernatant to another tube. The phosphorylation reaction commenced with the addition of a volume of the lysate supernatant (typically 250 µl) to a buffer containing 50 mM Tris, 5 mM ATP, 5 mM MgCl2, and 0.01 µCi/ml [14C]pantothenate (yielding a final pantothenate concentration of 0.2 µM). The solution had a pH of 7.4 and, unless specified otherwise, was prewarmed to 37 °C. At predetermined times, aliquots (200 µl) of the solution were transferred to a microcentrifuge tube containing 0.5 ml of 5.6% w/v PCA to halt the reaction. The tubes were centrifuged at 15,800 × g for 10 min and the resulting supernatant neutralized with 5% w/v KOH and processed as described above for the quantitation of [14C]pantothenate phosphorylation. Rates of phosphorylation were estimated from the slope of the initial, linear portion of the phosphorylation time courses.Estimate of Intracellular Water Volumes and Intracellular
Concentrations--
In all experiments with intact infected and
uninfected erythrocytes as well as saponin-permeabilized parasitized
erythrocytes, the intracellular water content of the cell pellets was
measured, thus enabling an estimate of the cell water volume and the
calculation of intracellular solute concentrations. The total water
volume of the pellet was estimated using 3H2O
content and the extracellular water volume estimated using either
[14C]inulin or [14C]sucrose. The
3H2O, [14C]inulin, and
[14C]sucrose were added to cells (at 0.5 µCi
ml1, 0.25 µCi ml
1, and 0.5 µCi
ml
1, respectively) 10 min before sampling. The
intracellular water volume was taken to be the difference between the
total and extracellular water volumes.
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RESULTS |
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Uptake and Phosphorylation of Pantothenic Acid by Intact Parasitized Erythrocytes-- Radiolabeled pantothenate was taken up rapidly into human erythrocytes infected with the mature (trophozoite) form of the malaria parasite (Fig. 2, open circles). The uptake time course was biphasic. There was an initial rapid influx (rate = 7.6 ± 1.5 µmol/(1012 cells · h); n = 3) lasting 1-2 min, during which time the intracellular concentration of radiolabel rose to 0.5-0.6 that in the external medium. This was followed by a second, slower phase (rate = 1.4 ± 0.3 µmol/(1012 cells · h); n = 3), during which the intracellular concentration of radiolabel increased (linearly with time) to levels well in excess of the extracellular concentration.
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Uptake and Phosphorylation of Pantothenic Acid by Saponin-permeabilized Parasitized Erythrocytes-- The data presented thus far provide clear evidence for the entry of pantothenate into malaria-infected erythrocytes via a furosemide-sensitive, nonsaturable pathway and its subsequent phosphorylation (a saturable process) within the parasitized cell. The question remains, however, of where in the cell the phosphorylation occurs.
In early work with duck erythrocytes infected with P. lophurae it was found that pantothenate kinase (5), as well as the other enzymes involved in the conversion of pantothenate to coenzyme A (6), were present in the cytosol of the duck erythrocyte but absent from the parasite, prompting the suggestion that conversion of pantothenate to coenzyme A takes place exclusively in the host cell cytosol. Quite different results were obtained here with P. falciparum. As shown in Fig. 6, parasites within saponin-permeabilized infected erythrocytes, lacking the enzymes of the red cell cytosol, accumulated radiolabeled pantothenate (in a predominantly phosphorylated form) from the medium. The maximal rate of accumulation of pantothenate by the parasites in this preparation, measured over the initial 30 min of the uptake time course, was 1.3 ± 0.3 µmol/(1012 cells · h) (n = 3). This value was not significantly different from the rate of accumulation of pantothenate by intact infected erythrocytes during the second phase of the biphasic uptake time course (i.e. 1.4 ± 0.3 µmol/(1012 cells · h); p = 0.74, unpaired t test).
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Phosphorylation of Pantothenate by Cell Lysates and Extracts-- The results obtained with saponin-permeabilized cells indicate that the intraerythrocytic form of P. falciparum has both a pathway for the transport of pantothenate across its plasma membrane and a pantothenate kinase. However, they do not necessarily identify the intracellular parasite as the site of phosphorylation of pantothenate within the intact parasitized cell. It remains possible that although the parasite possesses enzymes capable of phosphorylating pantothenate at the rate observed in intact parasitized erythrocytes, phosphorylation of pantothenate occurs primarily within the host erythrocyte cytosol, with the parasite taking up either fully formed coenzyme A or one of its phosphorylated precursors.
This possibility was addressed by comparing the relative [14C]pantothenate phosphorylation activities associated with the parasite and host cell compartments of parasitized cells, as well as that of uninfected erythrocytes, using a series of cell lysates and extracts (Fig. 7).
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DISCUSSION |
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Pantothenic acid is one of relatively few low molecular weight solutes for which the intraerythrocytic malaria parasite P. falciparum has an absolute extracellular requirement. Omission of pantothenic acid from the external medium results in cessation of parasite growth (2). The pathways involved in the transport and metabolism of this essential vitamin in parasitized cells therefore hold significant interest as chemotherapeutic targets.
Transport of Pantothenate into the Malaria-infected Erythrocyte-- In this study it was shown that pantothenate is impermeant to normal uninfected erythrocytes but enters malaria-infected erythrocytes rapidly via a pathway showing the characteristics (furosemide sensitivity, nonsaturability) of the NPP induced by the intracellular parasite in the host erythrocyte membrane. The NPP have been described previously in some detail (9-13). They have been shown to be permeable to a very wide range of low molecular weight solutes, including a number of nutrients required by the intracellular parasite. However, most of the latter compounds (e.g. glucose, nucleosides, and those amino acids either absent from or present at very low levels in the host cell hemoglobin) gain entry into the malaria-infected erythrocyte via endogenous host cell transporters; there are few, if any, essential nutrients for which there is clear evidence that their transport via the NPP plays a physiological role in the parasitized cell. In identifying the NPP as the sole route of entry of the essential vitamin, pantothenate, into the malaria-infected erythrocyte the present study provides evidence for a critical physiological role for the NPP. Furthermore, the finding that in the presence of furosemide the transport of the vitamin into the infected cell becomes rate-limiting for pantothenic acid utilization (Fig. 4) focuses attention on the possibility that the NPP may offer a suitable target for chemotherapeutic attack (20).
Metabolism of Pantothenate within the Parasitized Cell-- Once inside the infected erythrocyte, pantothenate is phosphorylated, the first step in its conversion to coenzyme A. Early work with P. lophurae-infected duck erythrocytes led to the proposal that pantothenate kinase (5) and the other enzymes involved in the conversion of pantothenate to coenzyme A (6) were located exclusively within the host erythrocyte compartment. However this is not the case in P. falciparum-infected human erythrocytes. Experiments with saponin-permeabilized parasitized erythrocytes demonstrated that the intracellular parasite has the ability to take up pantothenate from the extracellular solution (indicative of the presence of a pantothenate transport pathway at the parasite surface) and to phosphorylate it (indicative of the presence of a pantothenate kinase within the parasite).
The rate of accumulation of the phosphorylated form of pantothenate by parasites in saponin-permeabilized parasitized erythrocytes (1.3 ± 0.3 µmol/(1012 cells · h)) was similar to that measured in intact parasitized erythrocytes (1.4 ± 0.3 µmol/(1012 cells · h)). There is therefore sufficient activity within the parasite to account for most, if not all, of the phosphorylation activity observed within the intact infected erythrocyte. Experiments with cell extracts (carried out under conditions similar to those used in the earlier study of parasitized avian erythrocytes) revealed that although the host erythrocyte cytosol did contain a pantothenate kinase activity it was more than 10-fold less than that of the intracellular parasite and therefore insufficient to account for the phosphorylation activity observed in intact parasitized erythrocytes. Together, these results allow the conclusion that in P. falciparum-infected human erythrocytes most, if not all, of the phosphorylation of pantothenate, en route to coenzyme A, occurs within the intracellular parasite. The data do not permit the determination of whether the phosphorylation of pantothenate within the intact parasitized cell is mediated exclusively by the parasite (as might be the case if the NPP provide the pantothenate with a direct route to the parasite, without it passing through the erythrocyte cytosol (23)) or whether there is a minor contribution from the enzymes present in the host cell cytosol (as might be the case if, as has traditionally been assumed, solutes entering the cell via the NPP enter the red cell cytosol, from where they are taken up by the parasite (24)). Nevertheless, they do show conclusively that the model postulated for parasitized avian erythrocytes, in which the parasite is dependent upon the surrounding host cell cytosol for a supply of preformed coenzyme A is not applicable to P. falciparum-infected human erythrocytes.Conclusions-- Pantothenate is impermeant to normal human erythrocytes but is taken up rapidly into P. falciparum-infected erythrocytes via NPP induced by the parasite in the host cell membrane. Once inside the parasitized cell the pantothenate is phosphorylated, the first step in its conversion to coenzyme A. Under normal conditions the rate of transport of pantothenate into the infected cell is much faster than its subsequent phosphorylation. However, in the presence of an inhibitor of the NPP the initial transport step becomes rate-limiting for pantothenate utilization. Although the red cell cytosol does show low levels of pantothenate kinase activity, the majority of the phosphorylation occurs within the intracellular parasite. The enzymes involved are yet to be identified at a molecular level but these, together with the pantothenate transport pathways in both the parasite and host cell membranes, hold significant promise as targets for chemotherapeutic attack.
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ACKNOWLEDGEMENTS |
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We are grateful to Julie Kirk for assistance with data analysis.
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FOOTNOTES |
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* This work was supported by Australian National Health and Medical Research Council Grant 971008, Australian Research Council Grant F97082, the Ramaciotti Foundations, Wellcome Trust Grant 041182/Z/94/Z, and the Lister Institute of Preventive Medicine.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Lister Institute Research Fellow during the period that the initial experiments in this study were carried out. To whom correspondence should be addressed. Tel.: 61-2-6249-2284; Fax: 61-2-6249-0313; E-mail: kiaran.kirk{at}anu.edu.au.
1
The abbreviations used are: NPP, new
permeation pathways; PCA, perchloric acid; fl, femtoliters;
ATPS, adenosine 5'-O- (thiotriphosphate).
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REFERENCES |
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