Satiander Rana

Indian Institute of Integrative Medicine, Jammu City, Kashmir, India

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Publications (15)31.08 Total impact

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    ABSTRACT: Grewia asiatica L., an Asian shrub, is indigenous to India. Despite its immense importance in the Ayurvedic and traditional systems of medicine scientific and commercial exploration has been neglected. In this direction, we undertook systematic studies to understand the sexual system, breeding behaviour and reproductive phenology deployed by the species. Our results suggest that the species practices mixed mating as a consequence of temporal dichogamy of protandrous type and herkogamy with stigma slightly above stamens. Sequential floral evocation in acropetal order and profuse visitation by pollinators predispose G. asiatica to receive cross pollen. In a situation of non-receipt of pollen through insect pollinators, self-fertilization is prevalent by recurvation of receptive stigma towards dehiscent anthers. Seed set efficiency and fruit maturation are independent of pollen genotype (self/cross). Pre-emergent reproductive success (PERS), a measure of number of viable seeds that enter the ambient environment, was around 21%, which is indicative of moderate reproductive success. A self-compatibility index (SCI) and self-fertility index (SFI) showed values of 0.66 and 0.55, respectively, indicating the self-fertile and self-compatible nature of G. asiatica. The present study provides a detailed exposition of various reproductive features and breeding system prevalent in G. asiatica. These aspects are fundamental to understand the efficiency or failure of a species for genetic variation, crop improvement and evolutionary success.
    Flora - Morphology Distribution Functional Ecology of Plants 12/2014; · 1.46 Impact Factor
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    ABSTRACT: Background: Pharmacological investigations position withanolides as important bioactive molecules demanding their enhanced production. Therefore, oneof the pivotal aims has been to gain knowledge about complete biosynthesis of withanolides in terms of enzymatic and regulatory genes of the pathway. However, the pathway remains elusive at the molecular level. P450s monooxygenases play a crucial role in secondary metabolism and predominantly help in functionalizing molecule core structures including withanolides. Results: In an endeavor towards identification and characterization of different P450s, we here describe molecular cloning, characterization and expression analysis of two A-type P450s, WsCYP98Aand WsCYP76Afrom Withania somnifera. Full length cDNAs of WsCYP98Aand WsCYP76Ahave open reading frames of 1536 and 1545 bp encoding 511 (58.0 kDa) and 515 (58.7 kDa) amino acid residues, respectively. Entire coding sequences of WsCYP98A and WsCYP76AcDNAs were expressed in Escherichia coliBL21 (DE3) using pGEX4T-2 expression vector. Quantitative real-time PCR analysis indicated that both genes express widely in leaves, stalks, roots, flowers and berries with higher expression levels of WsCYP98Ain stalks while WsCYP76Atranscript levels were more obvious in roots. Further, transcript profiling after methyl jasmonate, salicylic acid, and gibberellic acid elicitations displayed differential transcriptional regulation of WsCYP98Aand WsCYP76A. Copious transcript levels of both P450s correlated positively with the higher production of withanolides. Conclusions: Two A-types P450 WsCYP98Aand WsCYP76Awere isolated, sequenced and heterologously expressed in E. coli. Both P450s are spatially regulated at transcript level showing differential tissue specificity. Exogenous elicitors acted as both positive and negative regulators of mRNA transcripts. Methyl jasmonate and salicylic acid resulted in copious expression of WsCYP98Aand WsCYP76A. Enhanced mRNA levels also corroborated well with the increased accumulation of withanolides in response to elicitations. The empirical findings suggest that elicitors possibly incite defence or stress responses of the plant by triggering higher accumulation of withanolides.
    BMC Biotechnology 11/2014; 14(89). · 2.59 Impact Factor
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    ABSTRACT: Rheum emodi (Polygonaceae), a multipurpose medicinal herb is a rich repository of pharmacologically active secondary metabolites known as anthraquinones. The present study entails HPLC quantitation and in vitro activity of four major constituents and the extracts of R. emodi. The anthraquinone glycosides were more abundant than their aglycone constituents viz. emodin and chrysophanol. MTT assay was used to assess the in vitro antiproliferative activity of anthraquinones and extracts on four cancer cell lines namely MIAPaCa-2, HCT-116, MCF-7 and T47D. The cytotoxicity was more obvious on MIAPaCa-2. Further, the study of mechanism of action involving cell cycle analysis and determination of mitochondrial membrane potential (MMP) loss was also investigated. The extracts significantly reduced cell viability by inducing apoptosis/necrosis and cell cycle arrest with concurrent loss of MMP (∆ψm) in a concentration dependent manner. The methanolic extract of rhizome (SPL5) with the least IC50 value (25 μg/ml) showed a significant increase in the percentage of apoptotic/necrotic cells (42.3% at 100 μg/ml) compared to that of vehicle treated cells (11.6%). These cellular manifestations corresponded remarkably with a decrease in integrity of the mitochondrial membrane. In conclusion, SPL5 with emodin and chrysophanol as the preponderant constituents exhibited considerable antiproliferative activity possibly by reducing cell viability and stirring up ∆ψm loss.
    South African Journal of Botany 08/2014; 95:1-8. · 1.34 Impact Factor
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    ABSTRACT: Picrorhiza kurrooa Royle ex Benth. is a highly reputed medicinal herb utilised in the preparation of a number of herbal drug formulations, principally due to the presence of novel monoterpene iridoid glycosides kenned as picrosides. Phenylalanine ammonia-lyase catalyses an important rate-limiting step in phenylpropanoid pathway and supplies precursors like cinnamic acid, vanillic acid, ferulic acid, etc., to a variety of secondary metabolites including picrosides. The imperilled status of P. kurrooa coupled with lack of information regarding biogenesis of picrosides necessitates deciphering the biosynthetic pathway for picrosides. In the present study, a PAL gene, designated PkPAL1 was isolated from P. kurrooa. The cDNA is 2312 bp in length, consisting of an ORF of 2142 bp encoding for a 713 amino acid protein having a predicted molecular weight of 77.66 kDa and an isoelectric point of pH 6.82. qRT-PCR analysis of various tissues of P. kurrooa showed that PkPAL1 transcript levels were highest in the leaves, consistent with picroside accumulation pattern. Using Genome walking, a 718 bp promoter region was also isolated resulting in identification of distinct cis-regulatory elements including TGA-element, TGACG motif, CGTCA-motif, etc. qRT-PCR indicated up-regulation of the PkPAL1 by methyl jasmonate, salicylic acid, 2, 4-dicholorophenoxy acetic acid and UV-B elicitations that corroborated positively with the identified cis-elements within the promoter region. Moreover, altitude was found to have a positive effect on the PkPAL1 transcript levels, driving the expression of PkPAL1 abundantly. Based on docking analysis, we identified eight residues as potentially essential for substrate binding in PkPAL1.
    Gene 06/2014; · 2.08 Impact Factor
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    ABSTRACT: Picrorhiza kurrooa synthesizes a large array of pharmacologically important monoterpenoid iridoid glycosides called picrosides. Although chemical profile and pharmacological activities of P. kurrooa have been extensively studied, limited attempts have been made to decipher the biosynthetic route and to identify the key regulatory genes involved in picroside biosynthesis. In the present study, NADPH–cytochrome P450 reductase, a key enzyme involved in electron transfer to cytochrome P450s was identified from P. kurrooa. The full length cDNA (2679 bp) contained an open reading frame of 2133 bp, corresponding to 710 amino acids. PkCPR was heterologously expressed in Escherichia coli and the kinetic parameters of the recombinant enzyme were determined. Specific activity, V max and K m of PkCPR were found to be 5.8 ± 0.05 μmol min−1 mg−1, 8.1 ± 0.12 μmol min−1 mg−1 and 7.8 μM, respectively. PkCPR was found to be spatially regulated at transcript level, being maximally expressed in leaf tissues. Altitude was found to have a positive effect on the picroside concentration and the picroside content positively correlated with the PkCPR transcript levels in samples collected at varied altitudes. Further, transcript profiling under methyl jasmonate, salicylic acid, 2,4-dicholorophenoxy acetic acid and UV-B elicitations displayed differential transcriptional regulation of PkCPR that fully corroborated with the identified cis-elements within the PkCPR promoter. Expression of PkCPR was inducible by UV-B and phytohormone elicitation, indicating that the PkCPR is possibly related to defence reactions, including biosynthesis of secondary metabolites. Present study is so far the only report of identification and functional characterization of CPR ortholog from P. kurrooa.
    Functional and Integrative Genomics 06/2014; · 2.69 Impact Factor
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    ABSTRACT: Oxidosqualene cyclases (OSCs) positioned at a key metabolic sub-dividing junction execute indispensable enzymatic cyclization of 2, 3-oxidosqualene for varied triterpenoid biosynthesis. Such branch-points present favourable gene targets for redirecting metabolic flux towards specific secondary metabolites. However, detailed information regarding the candidate OSCs covering different branches and their regulation is necessary for desired genetic manipulation. The aim of the present study, therefore, was to characterize members of OSC superfamily from Withania somnifera (Ws), a medicinal plant of immense repute known to synthesize a large array of biologically active steroidal lactone triterpenoids called withanolides. Three full length OSC cDNAs, β-amyrin synthase (WsOSC/BS), lupeol synthase (WsOSC/LS) and cycloartenol synthase (WsOSC/CS) having open reading frames of 2289 bp, 2268 bp and 2277 bp were isolated. Heterologous expression in Schizosaccharomyces pombe, LC-MS analyses and kinetic studies confirmed their mono-functionality. The three WsOSCs were found to be spatially regulated at transcriptional level with WsOSC/CS being maximally expressed in leaf tissue. Promoter analysis of three WsOSCs genes resulted in identification of distinct cis-regulatory elements. Further, transcript-profiling under methyl jasmonate (MeJA), gibberellic acid (GA3) and yeast extract (YE) elicitations displayed differential transcriptional regulation of each of the OSCs. Changes were also observed in mRNA levels under elicitations and further substantiated with protein expression levels by western blotting. Negative regulation by YE resulted in significant increase in withanolide content. Empirical evidence suggests that repression of competitive branch OSCs like WsOSC/BS and WsOSC/LS possibly leads to diversion of substrate pool towards WsOSC/CS for increased withanolide production.
    Journal of Biological Chemistry 04/2014; · 4.60 Impact Factor
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    ABSTRACT: Withania somnifera (L.) Dunal synthesizes large array of pharmacologically active secondary metabolites known as withanolides. It has been extensively investigated in terms of chemistry and bioactivity profiling. However, there exists fragmentary information about the dynamics of withanolide biosynthesis at different phenophases in concert with the expression analysis of key pathway genes. In the present study, two morpho-chemovariants of W. somnifera were harvested at five developmental stages, dissected into leaf and root tissues and assayed for three major withanolides viz. withanolide-A (WS-1), withanone (WS-2) and withaferin A (WS-3) content using high performance liquid chromatography. The present investigation also analyzed the expression pattern of five withanolide biosynthetic pathway genes namely squalene synthase, squalene epoxidase, cycloartenol synthase, cytochrome P450 reductase 1, cytochrome P450 reductase 2 to corroborate with the metabolite flux at different developmental stages. The relative transcript profiles of identified genes at various ontogenetic stages illustrated significant variation in leaf and root tissues and were largely concurrent with the alteration in withanolide pool. Comparatively, the concentrations of withanolide A, withanone and withaferin A along with expression levels of all the five genes were appreciably higher in the leaves than in roots. Relative dynamics in terms of quantitative and qualitative profiles of withanolides in leaf and root tissues revealed least correspondence between the pattern of accumulation, possibly indicting towards de novo tissue-specific biosynthesis.
    Molecular Biology Reports 11/2013; · 1.96 Impact Factor
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    ABSTRACT: Uridine diphosphate glycosyltransferases (UGTs) are pivotal in the process of glycosylation for decorating natural products with sugars. It is one of the versatile mechanisms in determining chemical complexity and diversity for the production of suite of pharmacologically active plant natural products. Picrorhiza kurrooa is a highly reputed medicinal herb known for its hepato-protective properties which are attributed to a novel group of iridoid glycosides known as picrosides. Although the plant is well studied in terms of its pharmacological properties, very little is known about the biosynthesis of these important secondary metabolites. In this study, we identified two family-1 glucosyltransferases from P. kurrooa. The full length cDNAs of UGT94F4 and UGT86C4 contained open reading frames of 1455 and 1422 nucleotides, encoding polypeptides of 484 and 473 amino acids respectively. UGT94F2 and UGT86C4 showed differential expression pattern in leaves, rhizomes and inflorescence. To elucidate whether the differential expression pattern of the two Picrorhiza UGTs correlate with transcriptional regulation via their promoters and to identify elements that could be recognized by known iridoid-specific transcription factors, upstream regions of each gene were isolated and scanned for putative cis-regulatory elements. Interestingly, the presence of cis-regulatory elements within the promoter regions of each gene correlated positively with their expression profiles in response to different phytohormones. HPLC analysis of picrosides extracted from different tissues and elicitor-treated samples showed a significant increase in picroside levels, corroborating well with the expression profile of UGT94F2 possibly indicating its implication in picroside biosynthesis. Using homology modeling and molecular docking studies, we provide an insight into the donor and acceptor specificities of both UGTs identified in this study. UGT94F2 was predicted to be an iridoid-specific glucosyltransferase having maximum binding affinity towards 7-deoxyloganetin while as UGT86C4 was predicted to be a kaempferol-specific glucosyltransferase. These are the first UGTs being reported from P. kurrooa. Copyright: ß 2013 Bhat et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
    PLoS ONE 09/2013; 8(9). · 3.53 Impact Factor
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    ABSTRACT: Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in withanolide biosynthesis. Present investigation so far is the only report of characterization of CPR paralogs from W. somnifera.
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    ABSTRACT: Withania somnifera (L.) Dunal, a highly reputed medicinal plant, synthesizes a large array of steroidal lactone triterpenoids called withanolides. Although its chemical profile and pharmacological activities have been studied extensively during the last two decades, limited attempts have been made to decipher the biosynthetic route and identification of key regulatory genes involved in withanolide biosynthesis. Cytochrome P450 reductase is the most imperative redox partner of multiple P450s involved in primary and secondary metabolite biosynthesis. We describe here the cloning and characterization of two paralogs of cytochrome P450 reductase from W. somnifera. The full length paralogs of WsCPR1 and WsCPR2 have open reading frames of 2058 and 2142 bp encoding 685 and 713 amino acid residues, respectively. Phylogenetic analysis demonstrated that grouping of dual CPRs was in accordance with class I and class II of eudicotyledon CPRs. The corresponding coding sequences were expressed in Escherichia coli as glutathione-S-transferase fusion proteins, purified and characterized. Recombinant proteins of both the paralogs were purified with their intact membrane anchor regions and it is hitherto unreported for other CPRs which have been purified from microsomal fraction. Southern blot analysis suggested that two divergent isoforms of CPR exist independently in Withania genome. Quantitative real-time PCR analysis indicated that both genes were widely expressed in leaves, stalks, roots, flowers and berries with higher expression level of WsCPR2 in comparison to WsCPR1. Similar to CPRs of other plant species, WsCPR1 was un-inducible while WsCPR2 transcript level increased in a time-dependent manner after elicitor treatments. High performance liquid chromatography of withanolides extracted from elicitor-treated samples showed a significant increase in two of the key withanolides, withanolide A and withaferin A, possibly indicating the role of WsCPR2 in withanolide biosynthesis. Present investigation so far is the only report of characterization of CPR paralogs from W. somnifera.
    PLoS ONE 02/2013; 8(2):e57068. · 3.53 Impact Factor
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    ABSTRACT: Withania somnifera is a rich reservoir of pharmaceutically active steroidal lactones known as withanolides. The plant is well characterized in terms of its chemistry and pharmacology, but very little is known about the pathway involved in the biosynthesis of withanolides. The present investigation describes the cloning, characterization and expression of squalene epoxidase (SE) gene from W. somnifera. SE (SQE; EC. 1.14.99.7) is one of the rate limiting enzymes in the biosynthesis of triterpenoids, catalyzing the stereospecific epoxidation of squalene to 2,3-oxidosqualene. A full length SE gene (WsSQE) of 1,956 bp was cloned which contained an open reading frame of 1,596 bp, encoding a protein of 531 amino acids with a predicted molecular mass of 57.67 kDa and theoretical PI of 8.48. Full length WsSQE was cloned into pGEX4T-2 vector and expressed in E.coli. Phylogenetic analysis indicated a significant evolutionary relatedness of WsSQE with squalene epoxidases of other plant species and the degree of relatedness with deduced amino acid sequences showed a significant correlation with different plant species. Using genome walking approach, a promoter sequence of 513 bp of WsSQE was isolated which revealed several key cis-regulatory elements known to be involved in various biotic and abiotic plant stresses. Comparative expression analysis of tissue specific WsSQE done by quantitative-PCR demonstrated the highest transcript levels in leaves, as compared to stalk and root tissues. This is the first report of cloning and bacterial expression of SE from W. somnifera and may be of significant interest to understand the regulatory role of SE in the biosynthesis of withanolides.
    Molecular Biology Reports 10/2012; · 1.96 Impact Factor
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    ABSTRACT: Withania somnifera, commonly known as ashwa-gandha or Indian ginseng, is a valuable medicinal plant, synthesizing a wide array of pharmacologically active second-ary metabolites known as withanolides. In this study, we investigated variation among 54 regenerated plants attained through indirect organogenesis from leaf explants. Organo-genic calli were induced on Murashige and Skoog medium containing 2 mgl −1 kinetin and 1 mgl −1 indole-3-butyric acid. High-performance liquid chromatography was used for quan-titative determination of the major withanolides in the soma-clones. One somaclone (WS-R-1) showed significantly higher accumulation of 12-deoxywithastramonolide (WS-12D; 0.516%) compared to the explant donor mother plant (0.002%). The incidence of somaclonal variation at the cyto-logical level was investigated by studying mitosis and meiosis in relation to chromosome number and structural organization. There were no alterations in chromosome phenotypes, somat-ic chromosome count, or meiotic behavior. Fidelity at ge-nomic level was evaluated by random amplification of polymorphic DNA (RAPD) analyses, which revealed multiple genetic polymorphisms between the WS-12D over-producing somaclone and the explant donor mother plant. This study demonstrates the capability of inducing chemotypic variabil-ity for the development of high-yielding clones due to molec-ular instability in W. somnifera using an in vitro approach.
    In Vitro Cellular & Developmental Biology - Plant 09/2012; · 1.16 Impact Factor
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    ABSTRACT: Picrorhiza kurroa Royle ex. Benth. is a medicinal herb of immense therapeutic value with restricted geographic distribution. Efficient plant regeneration via direct organogenesis and Agrobacterium tumefaciens-mediated genetic transformation was developed for this plant. Multiple shoot bud induction was achieved from leaf explants cultured in Gamborg’s B5 medium containing 3 % (w/v) sucrose, 3 mg/l kinetin and 1 mg/l indole-3-butyric acid. More than 90 % of leaf explants formed shoot buds leading to whole plant regeneration. An Agrobacterium-mediated genetic transformation protocol was developed using A. tumefaciens strain GV3101 harboring binary vector pCAMBIA1302 containing the green fluorescent protein and hygromycin phosphotransferase genes. Leaf explants precultured for 2 d were the most suitable for co-cultivation with Agrobacterium and transformation efficiency was enhanced with 200 μM acetosyringone. Putative transformants were selected using media containing 15 mg/l hygromycin. Transformation was verified by detection of the green fluorescent protein using fluorescence microscopy and by polymerase chain reaction. Approximately 56 % of the explants were transformed with an average of 3.4 ± 0.4 transgenic plantlets per explant. An efficient regeneration and transformation protocol thus developed enabling a fresh perspective of metabolic engineering in P. kurroa using an Agrobacterium-mediated transformation. This is the first report of direct organogenesis from leaf explants and genetic transformation of P. kurroa.
    In Vitro Cellular & Developmental Biology - Plant 06/2012; 48(3). · 1.16 Impact Factor
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    ABSTRACT: Withania somnifera (ashwagandha) is a rich repository of large number of pharmacologically active secondary metabolites known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, but there is sparse information about the genes responsible for biosynthesis of these compounds. In this study, we have cloned and characterized a gene encoding squalene synthase (EC 2.5.1.21) from a withaferin A rich variety of W. somnifera, a key enzyme in the biosynthesis of isoprenoids. Squalene synthase catalyses dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for sterols and triterpenes. A full-length cDNA consisting of 1765 bp was isolated and contained a 1236 bp open reading frame (ORF) encoding a polypeptide of 411 amino acids. Recombinant C-terminus truncated squalene synthase (WsSQS) was expressed in BL21 cells (Escherichia coli) with optimum expression induced with 1mM IPTG at 37°C after 1h. Quantitative RT-PCR analysis showed that squalene synthase (WsSQS) expressed in all tested tissues including roots, stem and leaves with the highest level of expression in leaves. The promoter region of WsSQS isolated by genome walking presented several cis-acting elements in the promoter region. Biosynthesis of withanolides was up-regulated by different signalling components including methyl-jasmonate, salicylic acid and 2, 4-D, which was consistent with the predicted results of WsSQS promoter region. This work is the first report of cloning and expression of squalene synthase from W. somnifera and will be useful to understand the regulatory role of squalene synthase in the biosynthesis of withanolides.
    Gene 03/2012; 499(1):25-36. · 2.20 Impact Factor
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    Journal of Plant Biochemistry and Biotechnology 01/2012; · 0.81 Impact Factor