Journal of Arid Environments xxx (2014) 1e7
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Snapshots of scorpion venomics Mohamed A. Abdel-Rahman a, b, *, Patrick L. Harrison b, Peter N. Strong b a b
Zoology Department, Faculty of Science, Suez Canal University, Ismailia 41522, Egypt Biomedical Research Center, Biosciences Division, Sheffield Hallam University, Sheffield, UK
a r t i c l e i n f o
a b s t r a c t
Article history: Received 8 May 2013 Received in revised form 20 January 2014 Accepted 22 January 2014 Available online xxx
Scorpions are particularly well adapted to survival in extreme habitats (especially arid and semi-arid environments) and their ability to produce and deliver venoms is an important factor in this success. Scorpion venoms are very complex mixtures of different proteins and peptides. Previous venomics studies revealed that each one of scorpion species may contain more than 100 different peptides. Scorpion venom peptides can be classified into two main types: disulfide-bridged peptides (DBPs) and non-disulfide-bridged peptides (NDBPs). The vast majority of DBPs are neurotoxic peptides that specifically interact with various types of ion-channels. The NDBPs have been shown to variously possess bradykinin-potentiating, antimicrobial, hemolytic, cellular signaling and immune-modulating activities. Recently, venom proteomics have been extensively applied in assessing the diversity of scorpion venom from various species. More insights about scorpion venom compositions were also gained through transcriptomic approach. It has provided an opportunity to obtain an overview of the content of scorpion venoms and to compare the relative abundance of toxin transcripts. More importantly, transcriptomics can reflect the biological processes occurring in venom gland cells. This review will highlight recent proteomic and transcriptomic studies to explore the venome of scorpions from different habitats, focusing on desert scorpions from North Africa. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Desert environment Proteomics Toxin peptides Transcriptomics Venome
1. Introduction: Scorpions and their natural habitats Scorpions are ancient animals and considered as one of the oldest known arachnids. According to Andrew (1990), the oldest fossil scorpions lived in the Paleozoic era 430 million years ago and these fossils are very similar to scorpions living today. Scorpions are belonging to class Arachnida which contains spiders, pseudoscorpions, mites and ticks. The main morphological differences between arachnids and insects are the absence of antennae and wings and the presence of 4 pairs of legs, rather than 3 pairs as in insects. When compared to spiders (about 39.000 species), scorpions are a modest group containing 1500 different species in 16 families (Fet et al., 2000). Of these families, the Buthidae is the largest (80 genera and over 800 species) and the most medically important (Soleglad and Fet, 2003). Scorpions are nocturnal animals, hiding during the day under stones, fallen wood and in other protected places. Some species seem to be attracted by human habitation (Abdel-Nabi et al., 2004; Abdel-Rahman et al., 2009; Anderson, 1983).
* Corresponding author. Zoology Department, Suez Canal University, Ismailia 41522, Egypt. E-mail addresses: [emailprotected], dr_moh_71@ hotmail.com (M.A. Abdel-Rahman).
Scorpions are recognized by the elongated body which is divided into three main parts, prosoma, mesosoma and metasoma (the latter, six segments containing the telson which holds a pair of venom glands). Scorpions have developed their venom gland as a sophisticated weapon used both in defending themselves against predators as well as to capture prey. Scorpions are widely distributed, most commonly associated with deserts in tropical and subtropical areas of the world. However they also occur in other varied habitats (e.g. forests, grassland, savannahs, intertidal zones, caves and even in freezing latitudes (Euscorpius flavicaudis, CloudsleyThompson and Constantinou, 1983). In order to cope with the extreme conditions of desert environments, scorpions have acquired a number of interrelated morphological, physiological and behavioral adaptations (Hadley, 1972). One of the major adaptations of many scorpion species in extreme environments is burrowing behavior. We (Abdel-Nabi et al., 2004) have found significant differences in burrow depths and shapes of the Egyptian burrowing scorpion Scorpio maurus palmatus (characterized by its large and bulky pedipalps) between scorpions collected from the arid desert of the Sinai Peninsula as compared to the semiarid Western Mediterranean Costal Desert (WMCD) (see Fig. 1). Burrows are deeper and more spiralled in arid localities than in wetter coastal areas.
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Several antimicrobial peptides (AMPs) (e.g. scorpine, pandinin, hadrurin, imcroporin, vejovine, BmKb1 and BmKn2) have been purified, primarily from Mexican, Chinese and South African scorpions (Conde et al., 2000; Corzo et al., 2001; Hernández-Aponte et al., 2011; Torres-Larios et al., 2000; Zeng et al., 2004). Although disulphide-bridged, defensin-like antimicrobial peptides from North African scorpions inhabiting the Sahara Desert (Androctonus, Leiurus) have been purified from scorpion haemolymph (EhretSabatier et al., 1996), no peptides from the corresponding venoms have been characterized. Using proteomic and genomic approaches, the venoms of scorpions living in the Sahara may therefore provide novel templates for the development of new antimicrobial agents. 3. Scorpion venomics
Fig. 1. Variation in the burrow shapes of the scorpion Scorpio maurus palmatus collected from different geographical regions in Egypt. Circles represent burrow entrances. W ¼ Western Mediterranean Costal Desert; E, R and S represent the three different Wadies of El-Agramia, Rahaba and Sahab in the desert of Southern Sinai, respectively (Abdel-Nabi et al., 2004).
2. Complexity of scorpion venoms Scorpion venom is a heterogeneous mixture of peptides, proteins, lipids, biogenic amines and nucleotides (Rodriguez de la Vega and Possani, 2005). Excluding the phenomena of intraspecific variation, each scorpion species may contain >100 peptide toxins (Possani et al., 2000). Biologically active peptides can be grouped into two main types (i) peptides with disulfide bridge(s) and (ii) peptides without a disulfide bridge (Catterall et al., 2007; Possani et al., 1999; Rodriguez de la Vega and Possani, 2005; Zeng et al., 2005). The majority of disulfide-bridged peptides are neurotoxins which specifically interact with various ion channels, including sodium, potassium, calcium and chloride channels (Dutertre and Lewis, 2010; Rjeibi et al., 2011; Rodriguez de la Vega and Possani, 2005; Valdivia and Possani, 1998). There are more than 250 fulllength amino acid sequences of scorpion neurotoxins that have been characterized and most of them are short peptides (23e76 amino acid residues constrained by 3 or 4 disulfide bridges). Some of these peptides selectively modulate mammalian ion channels while others specifically act on insect or crustacean channels. The venoms of dangerous scorpions (e.g. the desert scorpions Leiurus quinquestriatus, Androctonus australis, Androctonus crassicauda) primarily contain mammalian sodium channel toxins (60e76 amino acids cross-linked by 4 disulfide bridges) and potassium channel toxins (21e40 amino acids with 3 or 4 disulfide bridges) (Possani et al., 1999, 2000). Several short peptides that interact with either calcium or chloride channels have also been characterized (DeBin et al., 1993; Sidach and Mintz, 2002). Chlorotoxin (36-amino acids), purified from the venom of the desert scorpion L. quinquestriatus (DeBin et al., 1993), specifically blocks chloride channels of glioma cells. 131I-radiolabelled chlorotoxin has been used in clinical trials to treat glioma (Deshane et al., 2003), and chlorotoxin labeled with a far-infrared fluorescent imaging probe has been used to define the boundaries of glioma cells in vivo, suggesting its use in surgical tumor resection (Stroud et al., 2011). Peptides without a disulfide bridge are much smaller (13e50 amino acids). These peptides variously demonstrate hemolytic, cellular signaling, bradykinin-potentiating, immune-modulating and antimicrobial activities (Zasloff, 2002; Zeng et al., 2005).
It is estimated that animal venoms contain more than 20 million different peptides with a wide range of biological and pharmacological actions (Escoubas and King, 2009). Of these peptides, there are over 150,000 different peptides produced in the venom glands of approximately 1500 scorpion species from all over the world (Goudet et al., 2002; Rodriguez de la Vega et al., 2010). 828 scorpion venom peptides (or less than 0.2% of the estimated total) have been identified to date (ToxProt project, http://www.uniprot.org/ program/Toxins; Jungo et al., 2013). As illustrated in Fig. 2, the largest identified category (191 members) belongs to scorpion venom peptides of 60e70 amino acids residues. Consequently, it is important to explore the “Venome” (complete set of peptides and proteins expressed in a venom gland) of different scorpion species for several reasons: (1) to better understand the biodiversity of venoms and their molecular profiles, (2) to discover novel therapeutic and diagnostic agents; (3) to understand the complex pathophysiological effects of venoms on the prey of venomous animals and (4) to improve protection against scorpion envenomations through producing efficient antivenoms (Menez et al., 2005). Venomics (Escoubas et al., 2006) is a relevant approach to analyze animal venoms. It relies on a combination of mass spectrometry (proteomics) and molecular biology (genomics) methods. These techniques have been successfully applied to explore the “Venome” of scorpions and other venomous animals, including snakes (e.g. Calvete et al., 2007, 2009), spiders (e.g. Escoubas et al., 2006) and marine cone snails (e.g. Violette et al., 2012). In the following sections, recent proteomic and transcriptomic analyses of scorpion venom as well as venom glands will be outlined. 3.1. Proteomic analysis of scorpion venom Proteomic analysis of the whole scorpion venom is a very hard and challenging prospect (Vetter et al., 2011). In order to have an overview and estimate exactly how many unique structures (peptides/proteins) there are in a given scorpion venom, mass spectrometry techniques such as MALDI-TOF/MS and electrospray ionization (ESI) MS have been successfully applied. The second important step in any proteomic analysis is to determine the amino acid sequences of venom peptides (using Edman degradation or MS/MS) for main two reasons: (i) to study structureefunction relationships and (ii) to produce synthetic peptides for functional and structural studies. To date, detailed mass spectrometric proteomic studies of the venoms of 25 different scorpion species have been conducted (Fig. 3 and Table 1). Of these scorpions, fifteen (60%), are from the family Buthidae, five are from the family Scorpionidae, three are from the family Urodacidae and one each are from the families Hemiscorpiidae and Vaejovidae. The data in Table 1 clearly show the remarkable differences in the number of identified peptides between scorpion species, with a range of 60e665 different
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Fig. 2. Variation in the length (amino acids) of venom peptides characterized from various scorpion species (ToxProt project, 2013).
masses. The highest numbers of peptides were recorded in the Buthid venoms of Mesobuthus tamulus and Leiurus quinquestriatus hebraeus (665 and 554 peptides respectively), in comparison with the lowest, recorded from the venom of Tityus cambridgei (60 peptides). However mass fingerprinting of the 25 scorpion venoms revealed about 4826 different molecular masses (Fig. 3), few of these molecules were structurally and functionally characterized. The potential characterized peptides include sodium channel toxins (NaScTxs) such as Tc48a (Batista et al., 2004), Tst-3 (Batista et al., 2007), Tpa2 (Barona et al., 2006), Amm V, Amm VIII (Oukkache et al., 2008) and the potent mammalian a-sodium channel toxin Bu1 which has recently been purified from the
Turkish scorpion Buthacus macrocentrus (Caliskan et al., 2012). Another well characterized protein family is the potassium channel toxins (KScTxs) such as (i) butantoxin, TsKa, TsKb, TdK2, TdK3 Tpa1 and Tst-17 from scorpions inhabiting tropical and semi-arid environments of the Southern America (Barona et al., 2006; Batista et al., 2006, 2007; Diego-Garcia et al., 2005; Pimenta et al., 2001) and (ii) Amm TX3, Parabutoxin 1, Parabutoxin 2 are representative examples of KScTxs characterized from scorpions inhabiting the arid areas of Morocco and South Africa (Inceoglu et al., 2003; Oukkache et al., 2008) (Table 1). In addition to neurotoxins, various peptides have been assigned such as antimicrobial and bradykinin potentiating peptides (Diego-Garcia et al., 2005; Ma et al., 2010). Accordingly, the main advantages of using proteomic
Fig. 3. Differences in identified peptides, using proteomic analysis, in the venom of scorpions from different families represented as percentage (A) and total number of peptides.
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Table 1 Proteomic analysis of scorpion venoms collected from different habitats. Family
Scorpion species
Location/Habitat
Number and examples of potential identified toxins
References
Buthidae
Tityus serrulatus
Brazil/Tropical areas
Pimenta et al., 2001
Tityus cambridgei
Brazil/Tropical areas
Tityus costatus
Brazil/Atlantic Forest
Tityus stigmurus
Brazil/Semi-arid area
380 compounds detected using LC/ESI-MS and MALDI-TOFMS; Butantoxin, TsKa & TsKb (KScTxs) 60 compounds detected using ESI-MS and MALDI-TOFMS; Tc48a (NaScTxs) 90 compounds detected using LC/ESI-MS/MS; Butantoxin & scorpine- like peptides (KScTxs & AMPs, respectively). >100 unique masses were detected using MALDI-TOFMS; Tst-3, Tst-17 (NaScTxs & KScTxs, respectively). 464 compounds detected using 2D-LC/MS methods. 205 compounds detected using ESI-MS and MALDI-TOFMS; TdK2 & TdK3 (KScTxs) 104 compounds detected using ESI-MS/MS; Tpa1 & Tpa2 (KScTxs & NaScTxs, respectively). 380 compounds detected using 2D-LC/MS methods. 554 compounds detected using 2D-LC/MS.
Nascimento et al., 2006
80 compounds detected; Acra1 & Acra2 are toxic and lethal to mice, respectively. 70e80 compounds detected; Amm V, Amm VIII (NaScTxs) & Amm TX3 (KScTxs). 60 compounds detected; Bu1, a potent mammalian a- NaScTxs.
Caliskan et al., 2006.
Tityus bahiensis Tityus discrepans
Brazil/Atlantic Forest Venezuela/ Tropical areas Tityus pachyurus Colombia/ Tropical rainforest Leiurus quinquestriatus Sudan/ quinquestriatus Arid environment Leiurus quinquestriatus Middle-East/ hebraeus Arid environment Androctonus crassicauda Turkey/Semi-arid & arid environments Androctonus mauretanicus Morocco/ mauretanicus Arid environment Buthacus Turkey/Semi-arid macrocentrus & arid environments Parabuthus transvaalicus South Africa/Semi-arid & arid environments Mesobuthus India/Tropical coastal tumulus & semi-arid environments Lychas marmoreus obscurus Australia/Semi-arid & arid environments Urodacidae Urodacus Australia/Semi-arid yaschenkoi & arid environments Urodacus Australia/Semi-arid elongatus & arid environments Urodacus Australia/Semi-arid armatus & arid environments Scorpionidae Heterometrus longimanus Asia/Subtropical & tropical forests Pandinus South Africa/ cavimanus Tropical rainforest Heterometrus Asia/Subtropical petersii & tropical forests Opistophthalmus glabrifrons Africa, Forest & arid environments Egypt/Coastal Scorpio & arid environments maurus palmatus Hemiscorpiidae Opisthacanthus cayaporum Brazil/Open savannas Vaejovidae
Vaejovis spinigerus
North America/ Sonoran Desert
Batista et al., 2004 Diego-Garcia et al., 2005 Batista et al., 2007 Nascimento et al., 2006. Batista et al., 2006 Barona et al., 2006 Nascimento et al., 2006
Oukkache et al., 2008 Caliskan et al., 2012
100 compounds detected; Parabutoxin 1 & Parabutoxin 2 (KScTxs). Inceoglu et al., 2003 665 compounds detected using LC/ESI-MS and MALDI-TOFMS.
Newton et al., 2007
>100 compounds detected using MALDI-TOF matrix 1,5-DAN.
Smith et al., 2012
274 compounds detected using LCeMSeESI.
Luna-Ramírez et al., 2013
>100 compounds detected using MALDI-TOF matrix 1,5-DAN.
Smith et al., 2012
>100 compounds detected using MALDI-TOF matrix 1,5-DAN.
Smith et al., 2012
w78 compounds; ion channel inhibitors & AMPs.
Bringans et al., 2008
339 compounds detected using LC-MS/MS and MALDI-TOFMS.
Diego-Garcia et al., 2012.
w22 venom peptide families detected; a-KTxs, k- KScTxs, calcines and scorpine-like peptides. >100 compounds detected using MALDI-TOF matrix 1,5-DAN.
Ma et al., 2010
65 compounds detected using LCeMSeESI
Abdel-Rahman et al., 2013
221 compounds detected using LC/ESI-MS and by MALDI-TOFMS; scorpine- like peptide. >100 compounds detected using MALDI-TOF matrix 1,5-DAN.
Schwartz et al., 2008.
analysis are (1) to give a precise image about diversity (either interspecific or intraspecific) of scorpion venoms and (2) to open up the possibility of characterizing novel peptides that can potentially be developed into therapeutic agents. Whilst this proteomic approach has proved fruitful it is not without difficulties, mainly the large number of mass to charge ratio’s that are generated can make data analysis an arduous task. However if greater impetus is placed on the development of bioinformatics tools designed specifically to analyze this data then the speed and scope of venom research could be greatly improved. 3.2. Transcriptomic analysis of scorpion venom As discussed previously, most proteomic studies on scorpion venom have used milked venom. Recently, transcriptomic analysis has been employed for several reasons: (i) to gain further insights into venom diversity from both structural and functional
Smith et al., 2012
Smith et al., 2012
viewpoints, (ii) to understand biological processes taking place in the venom gland and (iii) to identify new venom peptide genes for more detailed mechanistic and genomic investigations (Zhang et al., 2006). This approach had primarily relied on the construction of cDNA libraries of venom glands, followed by random DNA sequencing of expressed sequence tags (ESTs; Fig. 4). ESTs analysis has successfully been applied to the venom glands of various venomous animals, fishes (Magalhaes et al., 2006), cone snails (e.g. Pi et al., 2006), snakes (e.g. Correa-Netto et al., 2011), spiders (Kozlov et al., 2005), and scorpions (e.g. Abdel-Rahman et al., 2013; Schwartz et al., 2007). The transcriptomes of seventeen different species of scorpion venom glands have been analyzed, resulting in the identification of more than 38,395 ESTs to date. This field is moving rapidly and the number of ESTs has increased ten-fold in the past twelve months (Fig. 5A). The vast majority of these (ca. 90%) have been obtained from the cloned venom glands of nine scorpions from the family Buthidae: Buthus occitanus Israelis
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Fig. 4. A simplified flowchart of scorpion venom gland transcriptome analysis. Scorpion venom glands were milked (1) using electrical stimulation 3e5 days before extraction total RNA (2) which used to prepare the full-length cDNA (3). The cDNA was digested (4) and the fractions ligated (5) into pDNR-LIB vector. Using electroporation, plasmids transformed (6) into DH5a and obtaining the cDNA library (7). Screening the transcriptome of venom gland through DNA sequencing (8) of colonies randomly selected and the high quality expressed sequence tags (ESTs) used for bioinformatic analyses (9-13).
(Kozminsky-Atias et al., 2008), Tityus discrepans (D’Suze et al., 2009), Lychas mucronatus (Ruiming et al., 2010), Hottentota judaicus (Morgenstern et al., 2011), Centruroides noxius (Rendon-Anaya et al., 2012), Tityus serrulatus (Alvarenga et al., 2012), Lychas mucronatus (Ma et al., 2012), Isometrus maculates (Ma et al., 2012), Tityus stigmurus (Batista et al., 2007). The remainder have been cloned from the venom glands of seven species of scorpion belonging to the families of Caraboctonidae (Hadrurus gertschi; Schwartz et al., 2007), Liochelidae (Opisthacanthus cayaporum; Silva et al., 2009), Euscorpiidae (Scorpiops jendeki and Scorpiops margerisonae; Ma et al., 2009, 2012), Scorpionidae (Heterometrus petersii, Pandinus cavimanus, Scorpio maurus palmatus; Ma et al., 2010; Diego-Garcia et al., 2012; Abdel-Rahman et al., 2013) and Urodacidae (Urodacus yaschenkoi; Luna-Ramirez et al., 2013) (Fig. 5B and C).
With the exception of H. judaicus and T. stigmurus, all transcriptomic studies mentioned above have used recently milked scorpion glands, involved in the process of venom regeneration, in order to get enriched toxin libraries. In the cases of H. judaicus and T. stigmurus, the cDNA libraries of these two scorpions have been produced from their “replete” venom glands (resting, or full venom glands). The transcriptome profiles of replete venom glands were significantly different from the profiles obtained from milked venom glands. These results with replete glands revealed new insights about the dynamics of transcriptional changes in scorpion venom glands (Almeida et al., 2012; Morgenstern et al., 2011). Possani’s group completed the first scorpion venom gland transcriptome analysis in 2007 (Schwartz et al., 2007) and subsequently were the first to apply the 454-pyrosequencing platform to perform global transcriptome analysis (Rendon-Anaya et al., 2012). Using
Fig. 5. ESTs obtained from the venom gland of different scorpion species. A, B and C represent comparison of ESTs according to period, scorpion families and species, respectively.
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this technique, 30,301 transcripts have been characterized from both the replete and milked venom glands of the Mexican scorpion Centruroides noxius. In addition to the new toxin families identified in the venom gland, 454-pyrosequencing revealed the presence of important features as small RNA and the microRNA processing machinery (Rendon-Anaya et al., 2012). Although, thousands of transcripts have been identified in the venom glands of various scorpion species and a large number of these sequences belong to known peptides (e.g. NaScTxs, KScTxs, antimicrobial and cytolytic peptides), there are still many new DNA sequences that still need to be characterized in order to verify their functions. We are extending our preliminary studies (Abdel-Rahman et al., 2013), of both transcriptomic and proteomic analyses of the Sahara desert scorpion, Scorpio maurus palmatus, which we confidentially expect will lead to the discovery of novel templates for drug discovery. 4. Conclusion Recent developments in analytical, separation and molecular approaches such as proteomics, transcriptomics and highthroughput screening have given us a plethora of techniques to explore the venomes of scorpions. With the help of these technologies, several hundred peptides as well as thousands of venom transcripts have been characterized from the venom glands of various scorpion species. Some of these peptides have been developed into therapeutic agents and as molecular probes to study the physiological functions of nervous and cardiovascular systems; however we still have work to do before a complete picture of scorpion venom peptide production and venom function is elucidated. There has been little attention given to the venomics (proteomics and transcriptomics) of North African scorpions inhabiting the Sahara desert. Here we have started to address this issue and indeed, is one of our main objectives in writing this report which encourage and promote toxinologists to focus on the understanding of toxins (including scorpion toxins) in desert environments. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments Dr. Mohamed A. Abdel-Rahman acknowledges travel grants from the TWAS-UNESCO Associateship Scheme (The World Academy of Sciences; Italy), the Arab Fund for Economic and Social Development (AFESD; The State of Kuwait) and the Daniel Turnberg UK/Middle East Travel Fellowship Scheme (The Academy of Medical Sciences, London) enabling him to study “Venomics” at the Institute of Biotechnology (National Autonomous University of Mexico; Mexico) and the Biomedical Research Centre (Sheffield Hallam University; UK). References Abdel-Nabi, I.M., McVean, A., Abdel-Rahman, M.A., Omran, M.A., 2004. Intraspecific diversity of morphological characters of the burrowing scorpion Scorpio maurus palmatus (Ehrenberg, 1828) in Egypt (Arachnida: Scorpionidae). Serket 9, 41e67. Abdel-Rahman, M.A., Omran, M., Abdel-Nabi, I.M., Ueda, H., McVean, A., 2009. Intraspecific variation in the Egyptian Scorpion Scorpio maurus palmatus venom collected from different biotopes. Toxicon 53, 349e359. Abdel-Rahman, M.A., Quintero-Hernandez, V., Possani, L.D., 2013. Venom proteomic and venomous glands transcriptomic analysis of the Egyptian scorpion Scorpio maurus palmatus (Arachnida: Scorpionidae). Toxicon 74, 193e207. Almeida Diego, D., Scortecci, K.C., Kobashi, L.S., Agnez-Lima, L.F., Medeiros, S.R.B., Silva-Junior, A.A., Junqueira-de-Azevedo, I.L.M., Fernandes-Pedrosa, M.F., 2012. Profiling the resting venom gland of the scorpion Tityus stigmurus through a transcriptomic survey. BMC Genomics 13, 362.
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