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. 2023 Jun 15;9(7):1303–1309. doi: 10.1021/acsinfecdis.3c00031

Dihydroartemisinin Disrupts Zinc Homeostasis in Plasmodium falciparum To Potentiate Its Antimalarial Action via Pyknosis

Hiroko Asahi †, Mamoru Niikura ‡, Shin-Ichi Inoue §, Fujiro Sendo ∥, Fumie Kobayashi ⊥, Akira Wada †,*
PMCID: PMC10353546  PMID: 37321567

Abstract

graphic file with name id3c00031_0005.jpg

Artemisinins have been used as first-line drugs worldwide to treat malaria caused by Plasmodium falciparum; however, its underlying mechanism is still unclear. This study aimed to identify the factors inducing growth inhibition via pyknosis, a state of intraerythrocytic developmental arrest, when exposing the parasite to dihydroartemisinin (DHA). Changes in the expression of genome-wide transcripts were assessed in the parasites treated with antimalarials, revealing the specific downregulation of zinc-associated proteins by DHA. The quantification of zinc levels in DHA-treated parasite indicated abnormal zinc depletion. Notably, the zinc-depleted condition in the parasite produced by a zinc chelator induced the generation of a pyknotic form and the suppression of its proliferation. The evaluation of the antimalarial activity of DHA or a glutathione-synthesis inhibitor in the zinc-depleted state showed that the disruption of zinc and glutathione homeostasis synergistically potentiated the growth inhibition of P. falciparum through pyknosis. These findings could help further understand the antimalarial actions of artemisinins for advancing malaria therapy.

Keywords: Plasmodium falciparum, dihydroartemisinin, zinc depletion, zinc homeostasis

The pathogenic parasite Plasmodium falciparum causes malaria, a serious infectious disease with 247 million cases and over 619,000 deaths globally in 2021, as estimated by the World Health Organization.1 Currently, malaria remains a major global health problem despite declining mortality rates over the past decade. Artemisinin and its derivatives (ARTs) are the first-line antimalarial drugs. The common structure of ARTs is a sesquiterpene lactone containing an endoperoxy bridge, and the scission of the endoperoxide is essential for their effective antimalarial activity.2,3 The scission reaction is facilitated by Fe(II) ions or heme in the parasites, and the resulting oxygen radicals produce a primary or secondary carbon-centered radical via rearrangement. The reactive radicals have been thought to alkylate biomolecules of parasites or to generate oxygen radical species,4,5 leading to intraerythrocytic developmental arrest and growth inhibition through oxidative stress. Recently, a variety of high-quality analytical studies have been conducted to identify ART targets.6−8 However, the efforts have not yet yielded a complete elucidation of the underlying mode of action. In addition, ART-resistant malaria strains9−11 have been found in Southeast Asia9,12 and Southern Africa.13 ARTs are used in combination with companion drugs to prevent the emergence of ART-resistant malaria and its worldwide infestation.11,14,15 In this context, elucidating the mode of action of ARTs is indispensable to finding new strategies to suppress the generation of ART-resistant strains and develop novel companion drugs for diversifying ART-based combination therapy.

Dihydroartemisinin (DHA) is a protonated form of artemisinin with potent antimalarial activity. In the previous study on the antimalarial action of DHA,16 we found that DHA condensed the chromatin in the early stage of intraerythrocytic development of P. falciparum to evoke a nuclear-shrunken state called “pyknosis”. In the process of growth inhibition along with pyknosis, we observed the downregulation of glutathione (GSH) reductase at the transcriptional level and a decrease in the concentration of GSH. The imbalance in GSH levels was a key phenomenon caused by DHA. However, based on the complexity of the multiple reactions of the radical species produced from DHA with biomolecules,4,5 the antimalarial action of DHA with pyknosis cannot be entirely explained by the disruption of GSH homeostasis.

In this study, to identify new molecular factors involved in the antimalarial action of DHA and the associated pyknosis in P. falciparum, we set strict criteria and conducted a comparative analysis of transcripts in response to antimalarial compounds such as DHA, 7(E)-benzylidenenaltrexone (BNTX), and rimonabant (RIMO). Based on the comparative gene expression profile analysis, DHA might likely cause a specific dysfunction of zinc-associated proteins in the early stage of intraerythrocytic development of P. falciparum. Hence, to clarify whether DHA induces zinc depletion, we quantitatively assessed the endogenous amount of Zn(II) ions after treating P. falciparum with DHA. Furthermore, to verify the disruption of zinc homeostasis as a key phenomenon that induces pyknosis and potentiates the antimalarial action of DHA, we evaluated the intraerythrocytic developmental arrest and growth inhibition of P. falciparum under the zinc-depleted conditions produced by a zinc chelator, N,N,N′,N′-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN), in the absence or presence of DHA or BNTX.

As previously reported, BNTX, an opioid receptor antagonist with glutathione-synthesis inhibitory effect, and RIMO, a cannabinoid receptor antagonist, were shown to induce pyknosis and exhibit moderate antimalarial activity.16 Moreover, after treating intraerythrocytic P. falciparum with DHA (Pf-DHA), BNTX (Pf-BNTX), or RIMO (Pf-RIMO), we quantified the expression of their transcripts and specifically revealed the downregulation of GSH reductase, suggesting it to be a molecular factor leading to pyknosis. To further explore other unidentified factors involved in the antimalarial activity of DHA, transcriptional data derived from Pf-DHA, Pf-BNTX, and Pf-RIMO were quantitatively and comprehensively compared (Figure S1). Based on control data obtained from P. falciparum grown in medium only (Pf-cont), less than 0.25-fold changes were observed in 7.1% of the Pf-DHA genome, 7.2% of the Pf-BNTX genome, and 6.8% of the Pf-RIMO genome. Meanwhile, greater than 4-fold changes were observed in 1.4% of the Pf-DHA genome, 2.6% of the Pf-BNTX genome, and 1.4% of the Pf-RIMO genome. Subsequently, among these genes, the transcripts whose quantitative alterations were more than two-folds between Pf-DHA and Pf-BNTX or Pf-RIMO were further analyzed (Table 1). Notably, we focused on GLO1, tGLO2, ZFP, ZIP1, ZIPCO, and CDF because their functions are closely associated with Zn(II) ions, and these enzymes and proteins account for 24% of the total extracted genes. GLO1 and tGLO217,18 have zinc active sites and cooperatively catalyze the conversion of toxic methylglyoxal to nontoxic d-lactic acid using GSH as a cofactor. ZFP19 forms a zinc-mediated structure and binds to particular DNAs as a transcriptional regulator. ZIP120 and ZIPCO21 are a zinc transporter and an iron and zinc transporter, respectively, that control intracellular zinc levels to maintain zinc homeostasis. Lastly, CDF22 is a cation transporter that decreases the concentration of Zn(II) ions by effusing cations ions out of the cytoplasm.

Table 1. Comparison of the Gene Expression in P. falciparum Induced by DHA, BNTX, or RIMO.

      Fold changeb
  
Gene name Gene ID of PF3D7a Signal in Pf-cont Pf-DHA Pf-BNTX Pf-RIMO Type of regulation in Pf-DHA
Glycosylation and glycolysis
(1) ALG14, putative _0211600 208 0.201 0.686 0.840 DR
(2) GLO1 _1113700 38 0.383 1.839 0.843 DR
(3) tGLO2 _1205700 77 0.204 0.426 1.089 DR
Host invasion
(4) PHISTc _0801000 210 2.092 0.966 0.330 UR
Lipid
(5) GDPD _1406300 1651 0.102 0.249 0.439 DR
Microtubule-based process
(6) Alpha tubulin 2 _0422300 152 2.085 1.039 0.289 UR
Mitochondrion
(7) QCR8, putative _0306000 184 0.122 1.088 0.492 DR
(8) BCKDHA, putative _1312600 163 2.650 0.740 0.157 UR
Nucleic acid binding and transcription
(9) KH1, putative _0605100 133 0.458 0.948 2.895 DR
(10) ZFP, putative _1019300 55 2.181 0.998 0.998 UR
Protein kinase
(11) S/T PK, putative _1338900 115 0.113 0.866 0.394 DR
Transmembrane transport
(12) MFSP, P115, putative _1117000 444 0.073 0.330 0.335 DR
(13) MFSP, MFS3, putative _0919500 55 0.203 0.966 0.621 DR
(14) ZIP1, putative _0609100 1869 0.269 0.589 1.099 DR
(15) ZIPCO, putative _1022300 23 0.220 0.348 0.480 DR
(16) CDF, putative _0715900 765 0.552 0.184 0.254 DR
Other
(17) PfEMP1, pseudogene _0500200 1105 0.137 1.475 0.316 DR
(18) IMP2, putative _0730400 123 0.169 0.891 1.093 DR
(19) Conserved Plasmodium membrane protein, UF _0718700 203 0.184 0.394 1.159 DR
(20) Conserved Plasmodium membrane protein, UF _1135300 338 0.229 0.610 1.015 DR
(21) Conserved Plasmodium protein, UF _0628700 619 0.246 0.492 1.271 DR
(22) Plasmodium exported protein, UF, pseudogene _1478400 2837 0.352 0.732 1.482 DR
(23) Conserved Plasmodium membrane protein, UF _0924500 1674 0.463 1.784 1.932 DR
(24) Plasmodium exported protein, UF _1038700 114 5.199 2.094 0.393 UR
(25) DnaJ protein, putative _1473200 125 9.070 1.251 2.755 UR
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a

The sequences of the selected transcripts were identified using the gene data of P. falciparum 3D7 strain (PF3D7), which was retrieved from PlasmoDB as a genomic database of P. falciparum and GenDB of the National Center for Biotechnology Information.

b

The expression levels of the transcripts of P. falciparum treated with DHA (Pf-DHA), BNTX (Pf-BNTX), or RIMO (Pf-RIMO) were quantitatively calculated on the basis of control data obtained from P. falciparum grown in medium only (Pf-cont). DR: downregulation, UR: upregulation, ALG14: UDP-N-acetylglucosamine transferase subunit ALG14, GLO1: glyoxalase I, tGLO2: targeted glyoxalase II, PHISTc: Plasmodium helical interspersed subtelomeric protein c, GDPD: glycerophosphodiester phosphodiesterase, QCR8: Cytochrome b-c1 complex subunit 8, BCKDHA: mitochondrial 2-oxoisovalerate dehydrogenase subunit alpha, KH: KH domain-containing protein, ZFP: zinc finger protein, S/T PK: serine/threonine protein kinase, MFSP: major facilitator superfamily domain-containing protein, ZIP1: zinc transporter ZIP1, ZIPCO: ZIP domain-containing protein, CDF: cation diffusion facilitator family protein, PfEMP1: P. falciparum erythrocyte membrane protein 1, IMP2: immune mapped protein 1-like protein, and UF: unknown function.

Because Zn(II) ions play essential roles in a wide variety of biological systems,23−26 the dysfunction of the identified zinc-associated enzymes and proteins would likely affect the intraerythrocytic development and growth cycle of P. falciparum. In particular, the alterations in the transcriptional levels of ZIP1, ZIPCO, and CDF would impact the transport of Zn(II) ions into and out of the cytoplasm. To ascertain whether DHA induced changes in the concentration of Zn(II) ions in parasites, zinc levels were quantified using a molecular reagent27 that fluoresces in response to binding to the Zn(II) ion (see Methods). As shown in Figure 1, an increase in zinc levels between 6 and 18 h was observed even in the parasites exposed to DHA. However, the amount of Zn(II) ions in DHA-treated parasites did not reach the same level as that in nontreated controls. Therefore, the phenomena suggest that the ability to uptake Zn(II) ions was drastically lowered due to the downregulation of ZIP1 and ZIPCO rather than CDF, resulting in abnormal zinc depletion.

Figure 1.

Figure 1

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Quantification of Zn(II) ions in P. falciparum treated with DHA. After culturing P. falciparum synchronized at the ring stage for 6 and 18 h in a medium with or without DHA (10 or 40 nM), the total amount of Zn(II) ions was quantified using a fluorescent molecule with specific zinc-binding affinity (as shown in bars and the left-hand vertical axis). The degree of parasitemia was calculated using the proportion of human red blood cells (RBCs) infected with P. falciparum, as observed through Giemsa staining (as depicted by diamond dots and the right-hand vertical axis). The data are described as the mean ± standard deviation (n = 3). The statistical analysis was performed using analysis of variance. *P < 0.05.

TPEN is a membrane-permeable zinc chelator in mammalian cells.28,29 To confirm whether TPEN can be used to effectively cause zinc depletion in P. falciparum, we examined its antimalarial activity depending on metal-binding specificity. As the concentration of TPEN increased, the growth rate of P. falciparum decreased (Figure S2A). The IC50 value of TPEN was calculated to be 1.8 ± 0.1 μM. Moreover, out of the supplementation trials with Zn(II), Fe(II), or Cu(II) ions (Figure S2B), only supplementary Zn(II) ions sufficiently suppressed the growth inhibitory effect of TPEN. Therefore, TPEN can specifically bind to the Zn(II) ion in P. falciparum, leading to a state of zinc depletion and subsequent growth inhibition.

To investigate the effect of TPEN-mediated zinc depletion on the intraerythrocytic development of P. falciparum, morphological changes in the presence of various concentrations of TPEN were monitored after synchronizing the parasites at the ring stage. As shown in Figure 2A, the absence of TPEN normally allowed for the developmental transition from the ring stage to the trophozoite and schizont stages. In contrast, increasing concentrations of TPEN gave rise to morphological changes from the ring to pyknotic forms. Furthermore, regardless of treatment with TPEN, normal developmental progress was restored as the amount of Zn(II) ions in the culture medium increased (Figure 2B). These phenomena show that only the depletion of Zn(II) ions in P. falciparum can induce pyknosis in the early stage of intraerythrocytic development, just as during exposure to DHA.

Figure 2.

Figure 2

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Effect of zinc depletion induced by TPEN on intraerythrocytic development of P. falciparum. P. falciparum, synchronized at the ring stage, was cultured for 24 h in the presence of (A) TPEN at various concentrations or (B) TPEN (32 μM) with graded supplementation of Zn(II) ions. The frequency of morphological changes was individually quantified by microscopically counting parasites of the pyknotic form (upper left), the ring stage (upper center), and the trophozoite plus schizont stages (upper right) after Giemsa staining. Pf-RBCs represent the human red blood cells infected with P. falciparum. The data are shown as the mean ± standard deviation (n = 3). The statistical analysis was performed using analysis of variance. *P < 0.05.

We previously proposed that pyknosis occurred when P. falciparum was treated with DHA; this was attributed to the disruption of GSH homeostasis following oxidative stress.16 To validate this hypothesis, the intraerythrocytic development of P. falciparum, synchronized at the ring stage, was assessed in the presence of DHA with N-acetyl-l-cysteine (NAC), which quenches various radical species, or supplemental GSH to compensate for a lack of endogenous GSH. Predictably, as concentrations of both NAC and GSH increased, the generation of the pyknotic form reduced, facilitating the progression of development to the trophozoite and schizont stages (Figures 3A-I and A-II). Therefore, the quenching of radical species and the maintenance of GSH levels significantly suppressed the induction of pyknosis by DHA, albeit not completely. Additionally, under the same conditions, an assessment was also performed in the presence of TPEN instead of DHA. NAC and GSH only slightly inhibited the emergence of pyknosis (Figures 3B-I and B-II). These observations indicate that zinc depletion can evoke pyknosis without generating radical species or disrupting GSH levels in P. falciparum.

Figure 3.

Figure 3

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Intraerythrocytic development of P. falciparum in the presence of DHA or TPEN supplemented with NAC or GSH. P. falciparum, synchronized at the ring stage, was cultured for 24 h in the presence of 50 nM DHA (A) or 20 μM TPEN (B) with graded supplementation of NAC (I) or GSH (II). The frequency of morphological changes was individually quantified by microscopically counting parasites of pyknotic form, ring form, and trophozoite plus schizont forms after Giemsa staining. Pf-RBCs represent the human red blood cells infected with P. falciparum. The data are shown as the mean ± standard deviation (n = 3). The statistical analysis was performed using analysis of variance. *P < 0.05.

To further clarify whether disruption of zinc homeostasis (Figure 1) correlates with the antimalarial activity of DHA, the combined growth inhibitory effect of DHA and TPEN was assessed. As shown in Figure 4A, DHA alone cannot fully inhibit the proliferation of P. falciparum owing to its low concentration. However, the combination of DHA and TPEN exhibited a markedly higher growth inhibitory effect than TPEN alone. This phenomenon indicates that zinc depletion generated by TPEN acts as a trigger to exert the antimalarial activity of DHA.

Figure 4.

Figure 4

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Growth inhibitory effect of TPEN against P. falciparum in combination with DHA (A) or BNTX (B). Asynchronous P. falciparum was cultured for 72 h in the presence of a low concentration of DHA (1 nM) or BNTX (1 μM) with TPEN at various concentrations. The growth rates were calculated based on the rate of changes in parasitemia. The data are shown as the mean ± standard deviation (n = 3). The statistical analysis was performed using analysis of variance. *P < 0.05.

Previously, we found that altered expression of GSH reductase in P. falciparum treated with BNTX was implicated in growth inhibition.16 Furthermore, in this study, we demonstrated that zinc depletion could induce pyknosis and subsequent growth inhibition independently of oxidative stress and GSH levels (Figure 3). Nonetheless, because the fact that Zn(II) ions essentially participate in maintaining the thiol redox balance in numerous living organisms,25,26 a close relationship possibly exists between these two pathways. Thus, we evaluated the growth inhibitory effect with a combination of the low concentration of BNTX and TPEN. In contrast to the sole use of BNTX, an augmentation of growth inhibition was observed as the amount of TPEN increased (Figure 4B). This result reveals that the depletion of both GSH and zinc levels cooperatively suppresses the growth of P. falciparum. Based on these findings, we concluded that DHA disrupts not only GSH homeostasis but also zinc homeostasis to potentiate its antimalarial action synergistically via pyknosis.

In this study, based on the newly set criteria, the comprehensive expression of transcripts in P. falciparum treated with DHA, BNTX, and RIMO was quantitatively compared. This analysis significantly revealed that DHA perturbed the gene expression of zinc-requiring enzymes and zinc-transporting proteins, indicating the effect of DHA on the trafficking and usage of Zn(II) ions. In particular, the transcriptional downregulation of ZIP1 and ZIPCO involved in the transport of Zn(II) ions was associated with the drop in the total concentration of Zn(II) ions after treatment with DHA. In addition, we successfully replicated the zinc-depleted state by exposing the parasites to TPEN, a membrane-permeable zinc chelator. The resulting zinc depletion was sufficient to induce a developmental transition from ring to pyknotic forms and suppress the growth capability. Furthermore, the presence of NAC or GSH can rescue the parasites against oxidative stress, thereby abrogating the induction of pyknosis by DHA, albeit incompletely. Meanwhile, the number of pyknotic forms by TPEN-mediated zinc deletion was nearly unaffected under similar conditions. Therefore, the pyknosis evoked by DHA, and the subsequent growth inhibition, is attributable to the disruption of GSH homeostasis in combination with zinc depletion. This interpretation is further supported by an augmentation of the growth inhibition by DHA and BNTX in a zinc-depleted state. Taken together, these data demonstrate that zinc depletion is involved in the antimalarial action of DHA by potentiating its growth inhibitory effect against P. falciparum following pyknosis. The characterization of DHA-driven disruption of zinc homeostasis in the malaria parasite provides valuable insight into the in-depth understanding of the antimalarial action of DHA, with the potential to inform the development of new drugs for advancing malaria therapy.14,15

Methods

Asynchronous and Synchronous Malaria Parasite Cultures

The FCR-3/FMG strain of P. falciparum (ATCC30932), which is sensitive to antimalarial drugs including artemisinin derivatives, was used in all experiments. As previously reported,30 the parasites were maintained in RPMI 1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% growth-promoting fraction from bovine plasma GF21 (FUJIFILM Wako Pure Chemical), 2 mM l-glutamine (Sigma-Aldrich, Merck), 25 mM 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (Sigma-Aldrich, Merck), 24 mM sodium bicarbonate (Invitrogen, Thermo Fisher Scientific), 25 μg/mL gentamicin (Sigma-Aldrich, Merck), and 0.15 mM hypoxanthine (Sigma-Aldrich, Merck). Human red blood cells (RBCs) were obtained from the Japanese Red Cross Society (No. R020021). The RBCs were washed and dispensed into 24-well culture plates at the hematocrit of 2%. P. falciparum-infected RBCs (Pf-RBCs) were cultured in a humidified atmosphere of 5% CO2, 5% O2, and 90% N2 at 37 °C. The percent of Pf-RBCs represented as parasitemia was initially adjusted to 0.1% for a subculture or 0.3% for assessment of the growth inhibitory effects of antimalarial compounds by adding uninfected RBCs of 2% hematocrit. Synchronization of P. falciparum at the ring stage was attained through two successive exposures to 5% (w/v) d-sorbitol (Sigma-Aldrich, Merck) separated by a 46-h interval.16 After the second sorbitol treatment, the remaining schizonts of P. falciparum and debris of RBCs were removed by isopycnic density centrifugation with 63% Percoll PLUS (GE Healthcare). Unless noted otherwise, the synchronous parasites, whose initial parasitemia was adjusted to 5.0–6.0%, were used to assess the intraerythrocytic developmental progress.

Assessment of the Intraerythrocytic Developmental Progress and Growth Rate of P. falciparum

Following the procedures described in the text, intraerythrocytic P. falciparum was cultured with or without antimalarial compounds and other required reagents for the indicated incubation period. After collecting samples from the culture medium at the different time points, thin smears of Pf-RBCs were prepared and stained with Giemsa reagent (Sigma-Aldrich, Merck). The developmental progress of the parasites was assessed by discriminating their forms on the smears using a bright field microscope (Nikon Corporation). Parasitemia was calculated by randomly counting >10,000 cells containing both Pf-RBCs and uninfected RBCs. The growth rates were calculated by dividing the final parasitemia of each sample by the initial parasitemia before treatment with antimalarial compounds. After plotting each growth rate, the IC50 values (the concentration required to inhibit parasite growth by 50% compared to compound-free controls) were extrapolated from concentration–response curves. The IC50 values of DHA (Sigma-Aldrich, Merck), BNTX (Sigma-Aldrich, Merck), and RIMO (Sigma-Aldrich, Merck) against asynchronous P. falciparum were 4.6 ± 0.2 nM, 1.7 ± 0.1 μM, and 1.8 ± 0.2 μM, respectively. All experiments were independently performed twice at different time points, and each data point in the figures represents the average of the values obtained from three measurements.

Measurement of the Concentration of Zn(II) Ions in P. falciparum

Intraerythrocytic P. falciparum, synchronized at the ring stage, was cultured for 6 and 18 h with or without DHA. After the degree of parasitemia reached approximately 7.5%, the RBCs and Pf-RBCs (1.44 × 109 cells) were harvested. The Pf-RBCs were lysed by adding more than 10 times the volume of pure water to the harvested cells, and then the parasites were isolated by centrifuging the lysates at 1,750g for 10 min at 4 °C. The isolated parasites were disrupted by three freeze–thaw cycles and resuspended in 400 μL chilled phosphate-buffered saline. After gently vortexing the solutions, the supernatants were isolated by centrifugation at 8,000g for 5 min at 4 °C. According to the manufacturer’s instructions, Zn(II) ions in the supernatants were quantified using the highly zinc-selective fluorescent molecule ZnAF-227 (GORYO Chemical). The concentration of Zn(II) ions was determined based on the standard curve prepared using solutions containing various concentrations of zinc sulfate.

Statistical Analysis

The significance of the differences between means was evaluated using multivariate analysis of variance (ANOVA). The calculations were performed using GraphPad PRISM 5 software (GraphPad Software). The P-value threshold was set to 0.05, and all pairwise comparisons were made using Bonferroni’s post hoc test.

Acknowledgments

This research was supported in part by the “Grant-in-Aid for Challenging Research (Pioneering)” (JP18H05358, to A.W.) and the “Grant-in-Aid for Scientific Research (C)” (JP23K06522, to A.W.) from the Japan Society for the Promotion of Science (JSPS). The authors thank the Japanese Red Cross Society for providing human red blood cells (R020021) and Dr. K. Sakamoto (RIKEN BDR) for helpful support and advice.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsinfecdis.3c00031.

  • Additional materials and methods, gene expression profile analysis, and growth rates of P. falciparum in the presence of TPEN (PDF)

Author Contributions

# H.A. and A.W. contributed equally to this work.

The authors declare no competing financial interest.

Supplementary Material

id3c00031_si_001.pdf (264KB, pdf)

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