1, ACMS can be either non-enzymati-cally converted into quinolinic acid QA, fuelling NAD biosynthesis, or transformed by the action of ACMS decarboxylase ACMSD, also known as picoli-nate
Trang 1e-semialdehyde decarboxylase in complex with
1,3-dihydroxyacetonephosphate suggests a regulatory
link between NAD synthesis and glycolysis
Silvia Garavaglia1, Silvia Perozzi1, Luca Galeazzi2, Nadia Raffaelli2 and Menico Rizzi1
1 DiSCAFF Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, University of Piemonte Orientale ‘A Avogadro’, Novara, Italy
2 Department of Molecular Pathology and Innovative Therapies, Section of Biochemistry, Universita` Politecnica delle Marche, Ancona, Italy
Introduction
In humans, tryptophan at a level that exceeds the basal
requirements for protein and serotonin synthesis is
oxi-datively degraded through the kynurenine pathway,
producing the highly unstable intermediate
a-amino-b-carboxymuconate-e-semialdehyde (ACMS) [1] As
shown in Fig 1, ACMS can be either non-enzymati-cally converted into quinolinic acid (QA), fuelling NAD biosynthesis, or transformed by the action of ACMS decarboxylase (ACMSD, also known as picoli-nate carboxylase; EC 4.1.1.45) into a-aminomuconic
Keywords
cerebral malaria; kynurenine pathway;
metal-dependent amidohydrolase; NAD
biosynthesis; neurological disorders
Correspondence
M Rizzi, DiSCAFF, University of Piemonte
Orientale, Via Bovio 6, 28100 Novara, Italy
Fax: +39 0321 375821
Tel: +39 0321 375712
E-mail: rizzi@pharm.unipmn.it
Database
The atomic coordinates and structure
factors of hACMSD have been deposited
with the Protein Data Bank (http://
www.rcsb.org) with accession codes 2wm1
and r2wm1, respectively
(Received 1 July 2009, revised 8 September
2009, accepted 10 September 2009)
doi:10.1111/j.1742-4658.2009.07372.x
The enzyme a-amino-b-carboxymuconate-e-semialdehyde decarboxylase (ACMSD) is a zinc-dependent amidohydrolase that participates in picolinic acid (PA), quinolinic acid (QA) and NAD homeostasis Indeed, the enzyme stands at a branch point of the tryptophan to NAD pathway, and deter-mines the final fate of the amino acid, i.e transformation into PA, com-plete oxidation through the citric acid cycle, or conversion into NAD through QA synthesis Both PA and QA are key players in a number of physiological and pathological conditions, mainly affecting the central ner-vous system As their relative concentrations must be tightly controlled, modulation of ACMSD activity appears to be a promising prospect for the treatment of neurological disorders, including cerebral malaria Here we report the 2.0 A˚ resolution crystal structure of human ACMSD in complex with the glycolytic intermediate 1,3-dihydroxyacetonephosphate (DHAP), refined to an R-factor of 0.19 DHAP, which we discovered to be a potent enzyme inhibitor, resides in the ligand binding pocket with its phosphate moiety contacting the catalytically essential zinc ion through mediation of
a solvent molecule Arg47, Asp291 and Trp191 appear to be the key resi-dues for DHAP recognition in human ACMSD Ligand binding induces a significant conformational change affecting a strictly conserved Trp–Met couple, and we propose that these residues are involved in controlling ligand admission into ACMSD Our data may be used for the design of inhibitors with potential medical interest, and suggest a regulatory link between de novo NAD biosynthesis and glycolysis
Abbreviations
ACMS, a-amino-b-carboxymuconate-e-semialdehyde; ACMSD, a-amino-b-carboxymuconate-e-semialdehyde decarboxylase; DHAP,
1,3-dihydroxyacetonephosphate; PA, picolinic acid; QA, quinolinic acid.
Trang 2acid-e-semialdehyde, which possibly collapses to
picoli-nic acid (PA) [2,3] Therefore, by competing with the
non-enzymatic synthesis of QA, ACMSD ultimately
controls the metabolic fate of tryptophan catabolism
along the kynurenine pathway, and is a medically
rele-vant enzyme in light of the important roles played by
QA and PA in physiological and pathological
condi-tions Indeed, QA is not only a key precursor of
NAD, but also a potent neurotoxin that acts by
acti-vating the N-methyl-d-aspartate subtype receptor
for glutamate [4] QA imbalance was reported to be
associated with a number of neurological disorders,
including a wide range of neuropsychiatric and
neuro-degenerative disease states, such as epilepsy,
Alzhei-mer’s and Huntington’s diseases [5] Conversely, PA
has been reported to prevent the neurotoxic effects of
increased QA in the rat central nervous system, sug-gesting that a highly regulated production of these metabolites is required for normal nervous function [6] Consistently, it has recently been shown that the enzyme is highly expressed in primary adult neurons but not in SK-N-SH neuroblastoma cells, with a per-fect correlation between the observed expression profile and the associated variation in QA and PA levels [7], and other investigations have clearly demonstrated that changes in ACMSD activity are readily reflected
by serum and tissue QA levels [8] Moreover, PA exhibits important immunomodulatory properties, being able to stimulate apoptosis [9], to efficiently interrupt the progress of human HIV-1 infection
in vitro [10], and to activate macrophages in pro-inflammatory processes [11] Most recently, abnormally high brain levels of PA have been reported in a mur-ine model of cerebral malaria, a frequently fatal com-plication of Plasmodium falciparum infection; in the same model, pharmacological reduction of PA levels was demonstrated to correlate with a better disease outcome [12,13] ACMSD is not only present in higher eukaryotes, but also in some micro-organisms, in which the enzyme plays a key role in both the trypto-phan to QA transformation and catabolism of 2-nitro-benzoic acid [14,15] Extensive biochemical and structural characterizations have been carried out on Pseudomonas fluorescens ACMSD (PfACMSD), lead-ing to the discovery that the enzyme is a member of the metal-dependent amidohydrolase superfamily fea-turing an (a⁄ b)8 TIM barrel fold [16–18] Biochemical and structural analysis of PfACMSD led to proposal
of a non-oxidative decarboxylation catalytic mecha-nism, unprecedented amongst known decarboxylases [18,19] The gene encoding human ACMSD (hA-CMSD) was identified few years ago [20], and very recently the existence of two isoforms originating by alternative splicing was demonstrated; although comparably expressed in various organs, only the hACMSD I isoform was reported to be enzymatically active and extensively characterized [3] hACMSD shares a high degree of sequence identity with PfACMSD (38%), with strict conservation of all resi-dues that are proposed to play a key role in catalysis and are involved in co-ordination of the catalytically essential zinc ion As no ACMSD structure with bound substrate or inhibitor has been reported so far from any source, understanding of the ACMSD catalytic mechanism is still incomplete In light of the reported ACMSD upregulation in the liver of streptozotocin-induced diabetic rats and the suppres-sion of such elevation following insulin injection [21,22], we decided to investigate the effect of
N
CH 2 CH COOH
NH 2
L-kynurenine
CHO COOH COOH
NH 2
Tryptophan
α-amino-β-carboxymuconate-ε-semialdehyde (ACMS)
α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase
(ACMSD)
CHO COOH NH2
α-aminomuconic acid-ε-semialdehyde (AMS)
N
COOH
COOH
Quinolinic acid
non enzymatic
NAD
Glutaryl CoA
CO2 + ATP
non enzymatic
N COOH
Picolinic acid
Fig 1 The reaction catalyzed by hACMSD in a metabolic context.
ACMS is derived from tryptophan degradation through the
kynure-ine pathway, and, depending on hACMSD activity, has various
metabolic destinies.
Trang 3glycolytic intermediates on the enzyme activity.
Interestingly, several phosphorylated glycolytic
intermediates were found to be strong inhibitors of
hACMSD, of which 1,3-dihydroxyacetonephosphate
(DHAP) was the most potent and was therefore
selected for our structural investigation Our results
provide the first structural image of an ACMSD in a
ligand-bound form, and may be used to assist the
struc-ture-based rational design of enzyme inhibitors with
potential medical interest
Results and Discussion
Overall quality of the model
The three-dimensional structure of hACMSD in
com-plex with DHAP was solved by molecular replacement
and refined at a resolution of 2.0 A˚ The monomer
present in the asymmetric unit contains 332 residues
out of 336, one zinc ion, one DHAP molecule, one
glyc-erol molecule and a total of 216 solvent molecules No
electron density is present for the last four residues at
the C-terminus The stereochemistry of the model has
been assessed using the program procheck [23] Ninety
per cent of the residues fall in the most favoured
regions of the Ramachandran plot, with Asn148 and
His269 falling in disallowed regions However, for both
residues, the excellent electron density map allowed us
to unambiguously assign the observed conformation
Residue Pro293 was recognized as the cis conformer
Overall structure
hACMSD shows a molecular architecture that closely
resembles that described for PfACMSD [18],
compris-ing a distorted (a⁄ b)8barrel domain and a small
inser-tion domain (Fig 2) hACMSD and PfACMSD can
indeed be superposed with an rmsd of 1.6 A˚ based on
326 Ca pairs hACMSD folds into 12 a-helices, 11
b-strands and connecting loops Residues 14-48 form
the small insertion domain that comprises a short
a-helix and a three-stranded anti-parallel b-sheet; the
remaining protein residues form the (a⁄ b)8 barrel
domain and a C-terminal extension that comprises two
short a-helices Functional hACMSD was previously
reported to be a monomer in solution [3] Consistently,
one molecule is present in the asymmetric unit in our
crystal, although a dimer can be observed in the
crys-tal lattice by applying the cryscrys-tallographic two-fold
axis PfACMSD was reported to be a dimer, with
subunits related by a dyad axis in the crystal, and a
mixture of monomeric and dimeric forms in solution
[18] Therefore, the available structural data suggest
that the minimal functional unit in the ACMSD enzyme is a monomer and the biological significance,
if any, of the loose dimer observed in the crystalline state remains to be established The overall structural organization observed in hACMSD confirms the previ-ous assignment of the enzyme to the metal-dependent hydrolase superfamily [17,18], whose members feature
by a structurally conserved TIM a⁄ b barrel fold [24] The significant structural conservation observed between hACMSD and PfACMSD extends to the peculiar small insertion domain, which may be consid-ered a unique trait of ACMSDs
The metal centre and the ligand binding site The hACMSD active site is located in a crevice on the protein surface at the C-terminal opening of the b-bar-rel (Fig 2), with a Zn2+ ion occupying the metal centre and coordinating, with a distorted trigonal bipyramidal geometry, the strictly conserved residues His6 (2.0 A˚), His8 (2.1 A˚), His174 (2.2 A˚), Asp291 (2.2 A˚) and the water molecule w1 (2.1 A˚) (Fig 3A) The DHAP binding site protrudes from the metal
N-ter C-ter
Fig 2 Ribbon representation of the overall structure of hACMSD The (a ⁄ b) 8 barrel domain is colored in light blue and the ACMSD-specific small insertion domain in blue The DHAP molecule and the protein residues involved in metal coordination are shown as sticks; the Zn 2+ ion and the solvent molecule bridging the metal centre to the ligand are shown as magenta and cyan spheres, respectively.
Trang 4centre into a small pocket delimited by residues Asp291, Trp191, Met195, Arg47 and the Phe294-Pro295-Leu296 amino acid stretch (Fig 3A,B) The ligand binds in an extended conformation, with its phosphate moiety located in the proximity of the zinc ion and with the hydroxymethylene group pointing toward Pro295 DHAP interacts with the catalytically essential Zn2+ through mediation of the metal-coordi-nating solvent molecule w1, which establishes two strong hydrogen bonds with the ligand O1 and O1P atoms at 2.5 and 2.8 A˚, respectively Moreover, the ligand is engaged in a number of stabilizing interac-tions with the protein milieu by contacting both pro-tein residues and solvent molecules In particular, the DHAP phosphate moiety establishes a salt bridge with Arg47 (distance of 3.0 A˚), an electrostatic interaction with the Zn2+ ion (closest distance of 4.1 A˚) and an extensive network of hydrogen bonds involving a set
of well-ordered solvent molecules O1P contacts the solvent molecule w268 (2.8 A˚), and O2P interacts with Trp191 (2.8 A˚) and O3P with w51 (2.6 A˚), which in turn contacts w119 (distance of 2.9 A˚), which contrib-utes to fixing the Arg47 orientation by establishing a hydrogen bond with its NH1 atom (at 2.8 A˚) The DHAP aliphatic chain is sandwiched between Trp191 and Asp291, with its carbonyl oxygen contacting, at a distance of 2.7 A˚, both Asp291 and the solvent mole-cule w1 Finally, the DHAP hydroxyl group is found
at 3.2 A˚ from Arg47, whose guanidinium group is held
in the observed conformation by an aromatic stacking interaction with Phe297
Implications for catalysis ACMSD belongs to the metal-dependent amidohydro-lase superfamily and catalyses a non-oxidative decar-boxylation reaction through a still not completely understood mechanism Extensive biochemical, struc-tural and spectroscopic investigations, mainly carried out on PfACMSD, led to the proposal of two possible alternative catalytic mechanisms [18], whose common feature involves formation of a tetrahedral intermedi-ate resulting from the nucleophilic attack of the metal-bound hydroxyl group onto the substrate, as observed
in other members of the amidohydrolase superfamily [24,25] However, as no structure of complexes with either the substrate, product or inhibitors had been reported, precise identification of the protein residues involved in ligand recognition and catalysis remained elusive
Although our structural data do not allow us to discriminate between the two alternative catalytic
His 174
Asp 291
w 1
DHAP
Arg 47
Leu 296
Trp 191
Pro 295
w 1
DHAP
w 268
w 119
w 51
Met 195
Phe 294
C 1
A
B
Fig 3 Close-up view of the active site in hACMSD (A) The metal
centre The Zn2+ ion and the coordinated solvent molecule are
shown as magenta and cyan spheres, respectively The strictly
con-served protein residues forming the metal coordinative sphere and
the ligand molecule are drawn as balls-and-sticks, with the DHAP
phosphorous in orange The Zn 2+ coordinative bonds are indicated
by dotted lines, together with the hydrogen bonds established by
DHAP with the metal-coordinating solvent molecule w1 The
por-tion of the 2F o )F c electron density map covering the DHAP
mole-cule is shown in blue at the 1.2 r level (B) The DHAP binding site
with crucial protein residues and solvent molecules engaged in
ligand recognition and stabilization are drawn as balls-and-sticks and
spheres, respectively The major interactions established between
the DHAP inhibitor and the protein milieu are indicated by dotted
lines.
Trang 5mechanisms proposed for ACMSD [21], the
hA-CMSD:DHAP complex represents the first structure of
an ACMSD in a ligand-bound form and can be used
to provide insights into catalysis In particular, the
residues involved in inhibitor binding can be suggested
to be important players for recognition of the
physio-logical substrate ACMS On the basis of our structure,
we propose that the strictly conserved Asp291 is a
fun-damental residue for catalysis, not only because it
con-tributes to Zn2+ coordination but also because of its
direct involvement in substrate binding (Fig 3A,B)
Indeed, as detailed above, Asp291 stabilizes DHAP
through interaction with the inhibitor carbonyl group,
and is therefore likely to demonstrate an equivalent
role on ACMS by possibly recognizing its aldehyde
group (Fig 1) Our observation is in agreement with
what was observed for PfACMSD, where a significant
increase in the KMvalue was observed when the
equiv-alent residue (Asp294) was mutated to alanine [16]
Another residue emerging as a key molecular
determi-nant for ligand recognition in hACMSD is Arg47
(Fig 3B) Its guanidinium group contacts both the
phosphate and the hydroxyl moieties present on
DHAP, and appears to be a major contributor to
ACMS recognition by stabilizing interactions with the
negatively charged carboxylic groups and with the
aldehydic portion of the physiological substrate A
third residue of relevance for efficient ligand
recogni-tion is Phe297 Indeed, in the hACMSD:DHAP
struc-ture, this residue fixes the orientation of Arg47, and is
‘edge-on’ oriented with respect to the DHAP aliphatic
chain with a shortest distance of 3.7 A˚ from C1
(Fig 3B) Such a conformation is compatible with
establishment of an aromatic hydrogen bond between
Phe297 and the double bonds present in ACMS
We carried out a careful comparison between the
hACMSD:DHAP complex and the ligand-free form of
PfACMSD after optimal structural superposition
Although no major conformational changes affecting
entire domains or extended portions of the protein
structure were detected, a significant change in the
ori-entation of a few residues was observed (Fig 4)
Indeed, upon ligand binding, a severe conformational
rearrangement takes place for the side chains of Met195
and Trp191, which move unidirectionally toward the
substrate-binding pocket and become engaged with the
bound ligand Trp191 shows the most pronounced
movement, which consists of a rotation around the v2
dihedral angle of about 95, allowing formation of a
hydrogen bond with the DHAP phosphate group This
switch between two alternative conformations suggests
that the Trp191–Met195 couple is the main active site
gating determinant controlling ligand admission
Conclusion
hACMSD appears to be an important enzyme control-ling the cellular levels of QA, PA and NAD As a consequence, modulation of hACMSD activity could
be of considerable relevance in certain therapeutic con-texts [2,26–30] The various neurological disorders in which severe imbalance in the kynurenyne pathway is seen include cerebral malaria [31], where an elevated level of the pro-inflammatory PA has been proposed to contribute to the development of this frequently fatal clinical manifestations of the disease [12,32] Moreover, this disease also features a significant depletion of the NAD+ level [31,33] We propose that hACMSD inhi-bition could result in alleviation of cerebral malaria symptoms by controlling both PA and NAD levels [7,34–36]
Recently, a direct link between NAD synthesis and diabetes has been reported [37], suggesting that an increased NAD level is a desirable condition to combat the disease Intriguingly, ACMSD was reported to be overexpressed in streptozotocin-induced diabetic rats
Trp 191
w 1
DHAP
Met 195
Fig 4 Conformational changes affecting ACMSD upon ligand bind-ing The image was obtained by optimal superposition of the hA-CMSD:DHAP and PfACMSD:ligand-free structures Protein residues are colored in white for hACMSD and in green for PfACMSD, with the DHAP ligand in yellow; the protein portion shown by a ribbon representation refers to hACMSD, as do the amino acid numbers The alternative conformations of the Trp–Met couple can be observed; the arrows indicate the unidirectional movement of the two protein residues upon ligand binding The dotted line highlights the hydrogen bond established between the DHAP phosphate moiety and Trp191 in the ligand-bound form of the enzyme.
Trang 6[21,22], and insulin injection was observed to suppress
such elevation Therefore, we propose that inhibition of
hACMSD should be explored as a possible novel
thera-peutic avenue for the treatment of diabetes In this
respect, the significant enzyme inhibition that was
found to be exerted by intermediates of glycolysis
(Table 1), a pathway imbalanced in diabetes, is
intrigu-ing Moreover, our structural data reveal that the
enzyme active site is well suited to efficiently bind
DHAP, a central glycolytic intermediate We are
there-fore tempted to speculate on a possible physiological
relevance of the observed modulation of hACMSD
activity by glycolytic intermediates that would imply a
novel regulatory role of the enzyme in energy
metabo-lism Indeed, robust aerobic glycolysis requires
signifi-cant NAD+ availability, which could be sustained by a
burst of de novo dinucleotide biosynthesis through
tryp-tophan degradation, an event that implies efficient
AC-MSD inhibition Therefore, hACAC-MSD would act as a
regulatory link between glycolysis and NAD synthesis
The structure of hACMSD in complex with DHAP
may used for the design of potent and highly selective
enzyme inhibitors that may prove to be of potential
interest to reduce life-threatening complications of
cerebral malaria, and as an important tool in
validat-ing our proposal of hACMSD as a novel drug target
for the treatment of diabetes and to investigate its
proposed novel regulatory role
Experimental procedures
Enzyme expression, purification and inhibition
studies
The expression vector pHIL-D2-ACMSDI constructed
pre-viously [3] was used as a template to amplify the ACMSD
gene, resulting in a C-terminal (His6) fusion protein The amplicon was cloned into the pHIL-D2 vector and the NotI-digested construct was used to transform Pichia pas-toris GS115 cells [3] Expression of the recombinant pro-tein was achieved as described previously [3] Purification was performed as described previously [3] with the follow-ing modifications The hydroxylapatite column was washed with 130 mm potassium phosphate buffer pH 7.0, 50 mm NaCl, and the recombinant protein was eluted with
300 mm potassium phosphate buffer pH 7.0, 50 mm NaCl The active fractions were pooled and directly applied to a HisTrap HP column equilibrated in 10 mm potassium phosphate buffer pH 7.0, 100 mm NaCl After extensive washing with the equilibration buffer, elution was per-formed with a linear gradient of imidazole from 0–0.3 m
in the same buffer The active fractions were pooled and diluted 10-fold with 50 mm potassium phosphate buffer, concentrated by ultrafiltration with a YM30 membrane (Millipore SpA, Milan, Italy) and used for the crystalliza-tion trials Using 400 mL of yeast culture, approximately
1 mg of homogeneuos ACMSD was obtained, with a spe-cific activity (1.3 unitsÆmg)1) and purification index (243-fold) similar to those of the protein without the His tag, and a higher overall yield (68%) [3] hACMSD activity was determined specrophotometrically, as described previ-ously [3] The effect of the tested molecules on the enzyme activity was investigated in the presence of 5 lm ACMS substrate
Crystallization and structure determination Crystals of hACMSD were obtained using the vapour dif-fusion technique in hanging drop A volume of 1 lL of res-ervoir solution containing 1 mm DHAP, 2% poly(ethylene glycol) (PEG 400), 0.1 m Na⁄ Hepes pH 7.5, 2.0 m ammo-nium sulphate, was mixed with the same amount of a protein solution at a concentration of 12.7 mgÆmL, and equilibrated against 500 lL of the reservoir solution, at
20C The crystals grew to a maximum length of 0.2 mm
in approximately 1 week The presence of DHAP was found to be essential for crystallization, and we were unable to grow crystals of hACMSD in a ligand-free form For X-ray data collection, crystals were quickly equili-brated in a solution containing the crystallization buffer and 20% glycerol as the cryo-protectant, and flash-frozen
at 100 K under a stream of liquid nitrogen Data up to 2.0 A˚ resolution were collected using the ID23-2 beamline
of the European Synchrotron Radiation Facility (Grenoble, France) An X-ray fluorescence scan performed on the hA-CMSD crystals using the same beamline, clearly indicated the presence of a Zn metal ion bound to the enzyme Anal-ysis of the diffraction data set allowed us to assign the crystal to the trigonal P3221 or P3121 space group, with cell dimensions a= b = 86.27 A˚ and c= 92.84 A˚, containing one molecule per asymmetric unit with a
Table 1 Effect of glycolytic intermediates on the activity of
hA-CMSD Inhibition values refer to the percentage inhibition exerted
by the indicated metabolite, relative to a reaction carried out in the
absence of any inhibitor (control) Experiments were performed at
37 C in triplicate in the presence of 5 l M ACMS substrate.
Metabolite
Metabolite concentration (m M )
Inhibition (%) Dihydroxyacetonephosphate 1
0.5 0.1
100 100 70
0.5 0.1
100 100 25
Trang 7corresponding solvent content of 50% Data were
pro-cessed using the program mosflm [38], and the ccp4 suite
of programs [39] was used for scaling Structure
deter-mination for hACMSD was performed by means of
the molecular replacement technique, using the
coordi-nates of a monomer of PfACMSD as the search model
(Protein Data Bank code 2HBV) [18] The program
amore [40] was used to calculate both cross-rotation
and translation functions in the 10–4 A˚ resolution
range The solution of the rotation function was used
to perform the translation function calculations in
both the P3221 and P3121 space groups A clear
solu-tion was obtained for the former only, allowing us to
unambiguously assign P3221 as the correct space
group The initial model was subjected to iterative
cycles of crystallographic refinement using the
pro-gram refmac [41], alternated with manual graphic
sessions for model building using the program o [42]
Approximately 7% of the randomly chosen reflections
were excluded from refinement of the structure and
used for the free R-factor calculation [43] The
pro-gram arp⁄ warp [44] was used for adding solvent
mol-ecules When the R-factor decreased to a value of 0.27
at 2.0 A˚ resolution, inspection of the electron density
map in the enzyme active site clearly revealed the
pres-ence of one molecule of DHAP that was consequently
manually modelled based on both the 2Fo)Fc and
Fo)Fc electron density maps The subsequent
crystal-lographic refinement converged to an R-factor and a
free R-factor of 0.19 and 0.24, respectively, with ideal
geometry Data collection and refinement statistics are
given in Table 2
Illustrations Figures were generated by using the program pymol [45] (http://www.pymol.org)
Acknowledgements
The authors would like to thank Dr Franca Rossi (DiSCAFF, University of Piemonte Orientale, Novara) for critical reading of the manuscript This work was supported by grants from the Regione Piemonte (Ricerca Scientifica Applicata 2004), Ministero dell’Is-truzione, dell’Universita´ e della Ricerca (Bando PRIN 2007) and the Compagnia di San Paolo – IMI (Torino, Italy) in the context of the Italian Malaria Network
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