• Users Online: 229
  • Print this page
  • Email this page


 
 
Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 9  |  Issue : 1  |  Page : 20-24

Antileishmanial activity of auranofin against Leishmania major in vitro


1 Department of Medical Parasitology, School of Medicine, Kashan University of Medical Sciences, Kashan, Iran
2 Department of Laboratory Science, School of Allied Medical Science, Kashan University of Medical Sciences, Kashan, Iran

Date of Submission30-Apr-2021
Date of Decision10-Nov-2021
Date of Acceptance11-Dec-2021
Date of Web Publication29-Mar-2022

Correspondence Address:
Dr. Mahdi Delavari
Department of Medical Parasitology and Mycology, School of Medicine, Kashan University of Medical, Sciences, Kashan
Iran
Login to access the Email id

Source of Support: None, Conflict of Interest: None


DOI: 10.4103/iahs.iahs_77_21

Rights and Permissions
  Abstract 


Aim: Despite the various studies done in the field of cutaneous leishmaniasis treatment, there is still no perfect and safe drug for definite treatment of this disease. Therefore, the quest to find an appropriate drug continues. Previous studies have shown that auranofin has anti-leishmanial activity; therefore, in this study; the effect of auranofin on Leishmania major was studied. Materials and Methods: Effectiveness of four concentrations (1, 2, 4, and 8 μg/ml) of auranofin on L. major was studied. After culturing amastigotes and promastigotes of the parasite, IC50 was calculated. The ability of auranofin to induce apoptosis in promastigotes was evaluated, and the degree of fragmentation of promastigotes DNA after treatment with auranofin was studied. Subsequently, the ultrastructural changes induced by treatment with auranofin in promastigotes were studied. Using the obtained results, IC50 of auranofin against amastigotes and promastigotes was calculated as 1.007 and 2.38 μg/ml, respectively. Findings: Showed that auranofin induce apoptosis in L. major. The highest rate of apoptosis (%80.1) occurred at the concentration of 8 μg/ml and also auranofin-induced fragmentation of DNA. Considerable changes occurred in the shape of body and free flagellum of the L. major promastigotes after treatment with auranofin. Conclusion: Based on the results, it can be concluded that auranofin has a considerable anti-leishmanial activity and additional studies in this field will be based on the results.

Keywords: Apoptosis, auranofin, fragmentation, leishmania major, ultrastructural


How to cite this article:
Olya ME, Delavari M, Arbabi M, Rasti S, Hooshyar H, Salimian M. Antileishmanial activity of auranofin against Leishmania major in vitro. Int Arch Health Sci 2022;9:20-4

How to cite this URL:
Olya ME, Delavari M, Arbabi M, Rasti S, Hooshyar H, Salimian M. Antileishmanial activity of auranofin against Leishmania major in vitro. Int Arch Health Sci [serial online] 2022 [cited 2022 Sep 29];9:20-4. Available from: http://www.iahs.kaums.ac.ir/text.asp?2022/9/1/20/341222




  Introduction Top


Leishmania is a genus of the flagellate protozoa and belongs to the Trypanosomatidae family which is a causative agent of leishmaniasis.[1] The vectors of these protozoa are the Phlebotomus sand flies in the ancient world and the Lutzomyia in the new world.[2] Cutaneous leishmaniasis is one of the clinical forms of leishmaniasis. This disease has been present in the most parts of the world and is characterized by cutaneous ulcers.[3] According to the WHO reports, 700,000 to one million new cases of cutaneous leishmaniasis occur annually.[4] Various chemical agents such as miltefosine, Paromomycin, Amphotericin B, liposomal amphotericin B, Allopurinol, and Mepacrine are used in the treatment of this disease, but due to the imperfect treatment, relapse of the disease, the toxicity of the usual drugs and remaining wound scar, researchers are still working hard to find the appropriate drug.[5],[6],[7] Auranofin is chemically combined with the formula [2, 3, 4, 6-tetra-o-acetyl-L-thio-b-D-glycopyranp-sato-S- (triethyl-phosphine) gold]. It can be used in human diseases and does not have toxic properties.[8],[9],[10] A combination of auranofin consists of gold, phosphine plus ligands of thiol.[11] Auranofin inhibits oxidative/reversible enzymes, such as thioredoxin reductase,[12],[13] oxidative/reductase enzymes, including important enzymes in the control of reactive oxygen species and prevents damage to DNA of various cells, such as cancer cells, T-cells, and parasites.[12],[13],[14] Oxidative/reductases are enzymes that strongly protect parasitic protozoa.[15],[16] In 1985, auranofin was first used to treat rheumatoid arthritis.[17] This drug reduces inflammatory reactions in the body and is used to treat rheumatoid arthritis to reduce pain and swelling.[18],[19] Furthermore, auranofin inhibits the signaling pathways of IKK/nuclear factor κB and reduces the regulation of the expression of the pro-inflammatory cytokines such as interleukin-1β and tumor necrosis factor-α.[20],[21] Studies have shown that auranofin is effective against giardia, especially metronidazole-resistant giardiasis.[16] In a similar study, the effect of auranofin on the inhibition of the glutathione reductase receptor in Schistosoma mansoni was evaluated.[22] Recent studies have shown that auranofin has an activity against Trypanosoma cruzi[23] Plasmodium falciparum,[8] Leishmania infantum,[24] S. mansoni[25] and Entamoeba histolytica.[26] The results of the research indicate that auranofin is active against Gram-positive organisms in vitro and in the human body, including the clinical strains of Streptococcus pneumoniae and Staphylococcus aureus.[27] Antifungal activity of auranofin has been detected against Candida albicans biofilms and Cryptococcus and Candida species.[28],[29] Considering the prevalence and complications of cutaneous leishmaniasis and the necessity of its treatment, the aim of this study was to survey the anti-leishmanial activity of auranofin against L. major in in vitro condition.


  Materials and Methods Top


Auranofin was purchased from Santa Cruz Biotechnology Company.

Leishmania major Iranian standard strain (MRHO/IR/75/ER) was cultured in RPMI 1640 containing 15% fetal bovine serum (FBS) () and antibiotic at 24 ± 2°C.

To evaluate the toxicity of different concentrations of the drug, their effect on the macrophage was measured 24 h after culture. MTT method was used for this purpose. J774 macrophage cell line was purchased from Iranian Pasture Institute and cultured in RPMI 1640 containing 10% FBS.[6] Macrophages were cultured in 96 well plates (1 × 105 cells/well), after 24 h, plate was washed and different concentrations (1, 10, 20, 30, 40, and 50 μg/ml) of auranofin were added to wells, then plate was incubated at 37°C with 5% CO2. After this time, 20 μl MTT (5 mg/ml) reagent was added per well, and plate was incubated for 4 h. Then, supernatant was removed and 100 μl DMSO (dimethyl sulfoxide) was added to each well and after 10 min, OD was read at 540 nm. The percentage of viability of the cell is calculated by following the formula:

[AT-AB]/[AC-AB] ×100

Where, AB, AC and AT are OD of blank well, negative control and treated cells, respectively.

Using the results of the MTT test and IC50, the SI index was calculated.

A 100 μl of the medium contained 106 leishmania promastigotes cultured in 24 well plates, and different concentrations of Auranofin (μg/ml) were separately added to the wells. Forty-eight hours after adding the auranofin, the number of parasites was counted. All of these experiments were repeated three times and the average of all three replicates was calculated. Furthermore, three wells without auranofin were considered as negative control. IC50 of auranofin was calculated after 48 h using GraphPad Prism5 software.

Macrophages were cultured on a glass coverslip in tissue culture 12-well plates (1 × 105 cells/well) (18) and incubated at 37°C with 5% CO2 for 24 h. Supernatants were removed after 24 h and 500 μl RPMI 1640 containing 107 promastigotes in the stationary phase were added to each well. In order to place the parasites inside macrophages, 24 h incubation was done. After this time, different concentrations of auranofin were added to each well and after 24 and 48 h, coverslips were separated and stained with Giemsa stain and amastigote number per macrophages was counted.

The Annexin V-FITC Apoptosis Detection Kit (Biovision; USA) was used. Twenty-four hours after treatment, promastigotes were collected and 500 μl of the bonding buffer was added to each sample. Further in the dark conditions, 5 μl of the Annexin-V and PI solution was added. After 5 min incubation at the room temperature and dark (42), the intensity of the color absorbed by the cells was measured by a flow-cytometric apparatus (BD FACSCanto II Flow cytometer). Finally, the results were analyzed by the special software (Flow jo Software).

To evaluate the fragmentation of DNA, Apoptotic DNA Ladder Detection Kit (abcam) was used. The promastigotes encountered with different concentrations of auranofin are collected after 48 h. According to the Kit protocol, DNA of treated leishmania was extracted and electrophoresed in 1.2% agarose gel containing 0.5 μg/ml ethidium bromides. Ethidium bromide-stained DNA was 48 hours after treatment to conduct topographic studies and investigate ultrastructural changes in L. major, scanning electron microscopy was used and images were taken. For this purpose, the parasites were washed in sodium cacodylate buffer pH 7.2 and preserve in a primary fixative (2.5% glutaraldehyde in 100 mM sodium cacodylate buffer) and then samples were fixed in 2% OsO4 in cacodylate buffer and then washed with distilled water and then immersed in ethanol 50%–70% for dehydration, then the samples were immersed in hexamethyl-disilazane and finally after coating with gold, images were taken by the scanning electron microscope.


  Results Top


The results showed that auranofin has good antileishmanial effect. After 24 and 48 h, viability percent of promastigotes and amastigotes decreased significantly (differences between groups were significant based on drug concentration and time), and these results were more evident at the concentration of 8 μg/mL [Figure 1] and [Figure 2]. According to the obtained results, the IC50 of auranofin after 24 h was calculated as 2.38 and 1.007 μg/ml against promastigotes and amastigotes, respectively.
Figure 1: Viability percent of amastigote and promastigote of L. major, 24 h after treatment with auranofin

Click here to view
Figure 2: Viability percent of amastigote and promastigote of L.major, 48h after treatment with auranofin

Click here to view


CC50 (50% cytotoxic concentration) was determined and then SI (selectivity index) was calculated by the following formula: SI = CC50 against j774 cell line/IC50 against amastigote. SI was 11.7 μg/ml.

Auranofin-induced apoptosis in promastigotes of L. major 48 h after treatment. Early and late apoptosis, necrosis, and live cell percent are presented in [Figure 3]. The highest percentage of induction of apoptosis was observed in the 8 μg/ml.
Figure 3: Flow cytometry anlysis. Promastigotes staining with Annexin V and Propidium Iodide after treatment with different concentrations of auranofin after 48 h

Click here to view


Electrophoresis of the extracted DNA from the treated L. major promastigotes showed that auranofin-induced fragmentation in the promastigotes. Fragmentation is quite evident and is lower at 1 μg/ml concentration than the other concentrations of auranofin, while in the untreated (control) group, the fragmentation has not occurred [Figure 4].
Figure 4: Gel electrophoresis of extracted DNA of L. major after treatment with different concentrations of auranofin. M: marker, a: without treatment, b: 1μg/ml, c: 2μg/ml d: 4μg/ml, e: 8μg/ml

Click here to view


Auranofin has caused a change in the shape of the body and free flagellum of the L. major promastigotes. In the control group, shape of promastigote was normal, but in treated sample, the changes were quite evident [Figure 5].
Figure 5: Changes in body shape and flagella of promastigote after adding auranofin in culture media. a: Control group (without auranofin), b: 2μg/ml of auranofin, c: 4μg/ml of auranofin, d: 8μg/ml of auranofin

Click here to view



  Discussion Top


Despite extensive research in the treatment of this disease, researchers have still failed to introduce an effective and nontoxic drug that has no adverse effects on the body.[30] Various herbal extracts, alone or in combination, as well as many chemical drugs, have been investigated for the treatment of this disease, but similar studies have continued to achieve this goal.[30],[31] In recent years, the use of gold compounds has been used to treat many diseases and against a wide range of microorganisms.[19],[32] Auranofin is a compound containing gold drug that was approved by the US Food and Drug Administration in 1985 for the treatment of rheumatoid arthritis.[16],[33] The results of this work showed that IC50 against promastigote stage was 2.38 μg/ml. In addition, the findings of this study showed that auranofin had an inhibitory effect on the amplification of intramacrophage amastigotes. The IC50 against the amastigotes stage was 1.007 μg/ml. Generally, the effect of this drug on both forms was time and dose dependent, so that with increased concentration and duration of effect, the growth rate of the parasite decreased. The SI index calculated in this study was 11.7 which indicated that the drug was a nontoxic drug. Flow cytometry test was performed using two colors of annexin-V and propidium iodide. Flow cytometric results showed that the highest apoptosis (80.1%) was induced by 8 μg/ml of auranofin after 48 h, this result indicates that cell death occurs in the induction of apoptosis and cell death has not occurred through necrosis. The highest incidence of necrosis was <2%, indicating that auranofin is an effective agent for the induction of apoptosis. The qualitative changes that occurred in L. major DNA due to the presence of auranofin were investigated in this study. The DNA of the parasites affected by auranofin was fragmented, indicating the destruction of the parasite and the formation of apoptotic fragments. A study conducted by Sharlow et al.(2014) showed that auranofin possesses inhibitory effects on the growth of promastigote and intracellular amastigotes and the EC50 obtained from the amastigotes of L. major and L. amazonnesis was calculated to be 0.07 and 0.27 μM, respectively.[34] Kaiser et al. in a study on Trypanosome brucei rhodesience using auranofin, it was concluded that the antiparasitic activity of auranofin against this protozoa is desirable and significant and this action is performed by inhibiting kappa B kinase and thioredoxin reductase (IC50 = 0.01 μm).[35] In another study, Tejman-Yarden et al. showed that auranofin has antiparasitic effects against Giardia lamblia (especially metronidazole resistant Giardia) by inhibiting TrxR.[16] In another study, da Silva et al. showed that auranofin has antiparasitic activity against T. cruzi.[23] Previous studies have shown that triple cytotoxic compounds of gold can cause cell death through apoptosis. Nevertheless, for most gold compounds, apoptosis is mainly caused by direct mitochondria damage and is not the result of a primary DNA lesion.[36],[37],[38] In this framework, the pivotal role of thioredoxin reductase has been considered as a possible target for gold compounds. The prevention of thioredoxin reductase activity may eventually lead to a change in mitochondrial membrane potential, Cytochrome c release, and subsequently an apoptotic event.[36]


  Conclusion Top


Auranofin has an anti-leishmanial activity, which was evident from the results obtained from various tests. Given the suitability of SI for drug use, auranofin can be a good choice for complementary and animal studies in the treatment of cutaneous leishmaniasis.

Financial support and sponsorship

The Vice Chancellor for Research of Kashan University of Medical Sciences, Kashan, Iran granted this study (project No. 95134). This research is a part of Msc thesis.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Kaufer A, Ellis J, Stark D, Barratt J. The evolution of trypanosomatid taxonomy. Parasit Vectors 2017;10:287.  Back to cited text no. 1
    
2.
Singh S. New developments in diagnosis of leishmaniasis. Indian J Med Res 2006;123:311-30.  Back to cited text no. 2
    
3.
Yanik M, Gurel MS, Simsek Z, Kati M. The psychological impact of cutaneous leishmaniasis. Clin Exp Dermatol 2004;29:464-7.  Back to cited text no. 3
    
4.
World Health Organization. Leishmaniasis Key Facts. (2020). Available from: https://www.who.int/news-room/fact-sheets/detail/leishmaniasis [Last accessed on 2020 Dec 23].  Back to cited text no. 4
    
5.
Croft SL, Seifert K, Yardley V. Current scenario of drug development for leishmaniasis. Indian J Med Res 2006;123:399-410.  Back to cited text no. 5
    
6.
Alrajhi AA, Ibrahim EA, De Vol EB, Khairat M, Faris RM, Maguire JH. Fluconazole for the treatment of cutaneous leishmaniasis caused by Leishmania major. N Engl J Med 2002;346:891-5.  Back to cited text no. 6
    
7.
Berman JD. Human leishmaniasis: Clinical, diagnostic, and chemotherapeutic developments in the last 10 years. Clin Infect Dis 1997;24:684-703.  Back to cited text no. 7
    
8.
Sannella AR, Casini A, Gabbiani C, Messori L, Bilia AR, Vincieri FF, et al. New uses for old drugs. Auranofin, a clinically established antiarthritic metallodrug, exhibits potent antimalarial effects in vitro: Mechanistic and pharmacological implications. FEBS Lett 2008;582:844-7.  Back to cited text no. 8
    
9.
Madeira JM, Renschler CJ, Mueller B, Hashioka S, Gibson DL, Klegeris A. Novel protective properties of auranofin: Inhibition of human astrocyte cytotoxic secretions and direct neuroprotection. Life Sci 2013;92:1072-80.  Back to cited text no. 9
    
10.
Fricker SP In: Sigel A, Sigel H (eds) Metal ions in biological systems: Metal ions and their complexes in medication, vol. 41, CRC Press, Copyright 2004 by Marcel Dekker, Inc.  Back to cited text no. 10
    
11.
Roder C, Thomson MJ. Auranofin: Repurposing an old drug for a golden new age. Drugs R D 2015;15:13-20.  Back to cited text no. 11
    
12.
Fan C, Zheng W, Fu X, Li X, Wong YS, Chen T. Enhancement of auranofin-induced lung cancer cell apoptosis by selenocystine, a natural inhibitor of TrxR1 in vitro and in vivo. Cell Death Dis 2014;5:e1191.  Back to cited text no. 12
    
13.
Marzano C, Gandin V, Folda A, Scutari G, Bindoli A, Rigobello MP. Inhibition of thioredoxin reductase by auranofin induces apoptosis in cisplatin-resistant human ovarian cancer cells. Free Radic Biol Med 2007;42:872-81.  Back to cited text no. 13
    
14.
Pessetto ZY, Weir SJ, Sethi G, Broward MA, Godwin AK. Drug repurposing for gastrointestinal stromal tumor. Mol Cancer Ther 2013;12:1299-309.  Back to cited text no. 14
    
15.
Hirt RP, Müller S, Embley TM, Coombs GH. The diversity and evolution of thioredoxin reductase: New perspectives. Trends Parasitol 2002;18:302-8.  Back to cited text no. 15
    
16.
Tejman-Yarden N, Miyamoto Y, Leitsch D, Santini J, Debnath A, Gut J, et al. A reprofiled drug, auranofin, is effective against metronidazole-resistant Giardia lamblia. Antimicrob Agents Chemother 2013;57:2029-35.  Back to cited text no. 16
    
17.
Kean WF, Kean IR. Clinical pharmacology of gold. Inflammopharmacology 2008;16:112-25.  Back to cited text no. 17
    
18.
Snyder RM, Mirabelli CK, Crooke ST. The cellular pharmacology of auranofin. Semin Arthritis Rheum 1987;17:71-80.  Back to cited text no. 18
    
19.
Madeira JM, Gibson DL, Kean WF, Klegeris A. The biological activity of auranofin: Implications for novel treatment of diseases. Inflammopharmacology 2012;20:297-306.  Back to cited text no. 19
    
20.
Tsai WB, Chung YM, Zou Y, Park SH, Xu Z, Nakayama K, et al. Inhibition of FOXO3 tumor suppressor function by betaTrCP1 through ubiquitin-mediated degradation in a tumor mouse model. PLoS One 2010;5:e11171.71.  Back to cited text no. 20
    
21.
Hu MC, Lee DF, Xia W, Golfman LS, Ou-Yang F, Yang JY, et al. IkappaB kinase promotes tumorigenesis through inhibition of forkhead FOXO3a. Cell 2004;117:225-37.  Back to cited text no. 21
    
22.
Kuntz AN, Davioud-Charvet E, Sayed AA, Califf LL, Dessolin J, Arnér ES, et al. Thioredoxin glutathione reductase from Schistosoma mansoni: An essential parasite enzyme and a key drug target. PLoS Med 2007;4:e206.4  Back to cited text no. 22
    
23.
da Silva MT, Silva-Jardim I, Portapilla GB, de Lima GM, Costa FC, Anibal Fde F, et al. In vivo and in vitro auranofin activity against Trypanosoma cruzi: Possible new uses for an old drug. Exp Parasitol 2016;166:189-93.  Back to cited text no. 23
    
24.
Ilari A, Baiocco P, Messori L, Fiorillo A, Boffi A, Gramiccia M, et al. A gold-containing drug against parasitic polyamine metabolism: The X-ray structure of trypanothione reductase from Leishmania infantum in complex with auranofin reveals a dual mechanism of enzyme inhibition. Amino Acids 2012;42:803-11.  Back to cited text no. 24
    
25.
Angelucci F, Sayed AA, Williams DL, Boumis G, Brunori M, Dimastrogiovanni D, et al. Inhibition of Schistosoma mansoni thioredoxin-glutathione reductase by auranofin: Structural and kinetic aspects. J Biol Chem 2009;284:28977-85.  Back to cited text no. 25
    
26.
Debnath A, Parsonage D, Andrade RM, He C, Cobo ER, Hirata K, et al. A high-throughput drug screen for Entamoeba histolytica identifies a new lead and target. Nat Med 2012;18:956-60.  Back to cited text no. 26
    
27.
Aguinagalde L, Díez-Martínez R, Yuste J, Royo I, Gil C, Lasa Í, et al. Auranofin efficacy against MDR Streptococcus pneumoniae and Staphylococcus aureus infections. J Antimicrob Chemother 2015;70:2608-17.  Back to cited text no. 27
    
28.
Siles SA, Srinivasan A, Pierce CG, Lopez-Ribot JL, Ramasubramanian AK. High-throughput screening of a collection of known pharmacologically active small compounds for identification of Candida albicans biofilm inhibitors. Antimicrob Agents Chemother 2013;57:3681-7.  Back to cited text no. 28
    
29.
Fuchs BB, RajaMuthiah R, Souza AC, Eatemadpour S, Rossoni RD, Santos DA, et al. Inhibition of bacterial and fungal pathogens by the orphaned drug auranofin. Future Med Chem 2016;8:117-32.  Back to cited text no. 29
    
30.
Minodier P, Parola P. Cutaneous leishmaniasis treatment. Travel Med Infect Dis 2007;5:150-8.  Back to cited text no. 30
    
31.
Nagle AS, Khare S, Kumar AB, Supek F, Buchynskyy A, Mathison CJ, et al. Recent developments in drug discovery for leishmaniasis and human African trypanosomiasis. Chem Rev 2014;114:11305-47.  Back to cited text no. 31
    
32.
Colotti G, Ilari A, Fiorillo A, Baiocco P, Cinellu MA, Maiore L, et al. Metal-based compounds as prospective antileishmanial agents: Inhibition of trypanothione reductase by selected gold complexes. ChemMedChem 2013;8:1634-7.  Back to cited text no. 32
    
33.
Messori L, Marcon G. Gold complexes in the treatment of rheumatoid arthritis. Met Ions Biol Syst 2004;41:279-304.  Back to cited text no. 33
    
34.
Sharlow ER, Leimgruber S, Murray S, Lira A, Sciotti RJ, Hickman M, et al. Auranofin is an apoptosis-simulating agent with in vitro and in vivo anti-leishmanial activity. ACS Chem Biol 2014;9:663-72.  Back to cited text no. 34
    
35.
Kaiser M, Mäser P, Tadoori LP, Ioset JR, Brun R. Antiprotozoal activity profiling of approved drugs: A starting point toward drug repositioning. PLoS One 2015;10:e0135556.  Back to cited text no. 35
    
36.
Coronnello M, Mini E, Caciagli B, Cinellu MA, Bindoli A, Gabbiani C, et al. Mechanisms of cytotoxicity of selected organogold (III) compounds. J Med Chem 2005;48:6761-5.  Back to cited text no. 36
    
37.
Wang Y, He QY, Sun RW, Che CM, Chiu JF. GoldIII porphyrin 1a induced apoptosis by mitochondrial death pathways related to reactive oxygen species. Cancer Res 2005;65:11553-64.  Back to cited text no. 37
    
38.
Bindoli A, Rigobello MP, Scutari G, Gabbiani C, Casini A, Messori L. Thioredoxin reductase: A target for gold compounds acting as potential anticancer drugs Coordination. Chem Rev 2009;253:1692-707.  Back to cited text no. 38
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

Top
 
  Search
 
    Similar in PUBMED
   Search Pubmed for
   Search in Google Scholar for
 Related articles
    Access Statistics
    Email Alert *
    Add to My List *
* Registration required (free)  

 
  In this article
Abstract
Introduction
Materials and Me...
Results
Discussion
Conclusion
References
Article Figures

 Article Access Statistics
    Viewed1610    
    Printed56    
    Emailed0    
    PDF Downloaded131    
    Comments [Add]    

Recommend this journal