Life Sciences 109 (2014) 1–7

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Glucose modulates Pax6 expression through the JNK/p38 MAP kinasepathway in pancreatic beta-cells

Sivasangari Balakrishnan, Mohanraj Sadasivam, Arun Kannan,Antojenifer Panneerselvam, Chidambaram Prahalathan ⁎Department of Biochemistry, Bharathidasan University, Tiruchirappalli 620 024, India

⁎ Corresponding author. Tel.: +91 431 2407071 484.E-mail address: prahalath@gmail.com (C. Prahalathan

http://dx.doi.org/10.1016/j.lfs.2014.06.0090024-3205/© 2014 Elsevier Inc. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:

Received 3 March 2014Accepted 7 June 2014Available online 20 June 2014

Keywords:Pax6GlucoseJNKp38 MAP kinasePTENBeta-cells

Aim: The paired and homeodomain-containing transcription factor, paired box 6 (Pax6), has shown to playpivotal roles in beta-cell function, including cell survival, insulin biosynthesis and secretion. The present studyinvestigates the signaling events that regulate the modulation of Pax6 expression by glucose and the role ofthis modulation in cell survival in rat insulinoma-1E (INS-1E) cells.Main methods: INS-1E cells were incubated on 1 mM (low) or 25 mM (high) glucose overnight. To elucidate thesignaling pathways that regulate Pax6 expression, we utilized specific inhibitors. The siRNA transfection of Pax6into INS-1E cells was performed by electroporation. ThemRNA and protein levels were determined by real-timePCR and Western blotting, respectively.Key findings:We found that the mRNA and protein levels of Pax6 were reduced by approximately 4-fold in high,compared to low, glucose-treated cells. Staurosporine, the c-Jun N-terminal kinase (JNK) inhibitor SP600125 andthe p38 mitogen-activated protein kinase (p38 MAPK) inhibitor SB203580 significantly increased Pax6 levels in

high glucose-treated INS-1E cells compared to their respective controls. However, neither calcium ionophore northe extracellular signal-regulated kinase (ERK) inhibitor U0126 resulted in any alteration in Pax6 protein expres-sion. Further, a siRNA-mediated knockdown of Pax6 significantly decreased the expression of tumor-suppressorphosphatase with tensin homology (PTEN) while increasing cell viability in low glucose-treated INS-1E cells.Significance: This study addresses the signaling events that regulate the glucose-dependent expression of Pax6and the role of these events in cell survival in pancreatic beta cells.

© 2014 Elsevier Inc. All rights reserved.

Introduction

Glucose homeostasis is principally maintained by the alpha and betacells of pancreatic islets through the precisely regulated release ofglucagon and insulin, respectively. Glucose coordinately recruits a high-ly sophisticated network of transcription factors and co-activators to theinsulin promoter and controls insulin production and secretion. In addi-tion to insulin, glucose regulates the expression of various genes thatencode the proteins involved in cellular maintenance, repair, transcrip-tion and RNA splicing. The glucose regulation of beta-cell function is animportant mechanism by which cells can adapt their metabolismand function to variations in the concentration of this vital nutrient(Poitout et al., 2006; Meugnier et al., 2007; Martinez et al., 2006).

Paired box 6 (Pax6) is a transcription factor with two DNA-bindingdomains (a paired box and a homeobox) and a proline–serine threonine(PST)-rich transactivation domain at the C terminus (Mishra et al.,2002). Pax6 plays a vital role in the development of the eye, central

).

nervous system and the pancreas (Kozmik, 2008; Sansom et al., 2009;Hart et al., 2013) and is crucial for beta cell maturation through its tran-scriptional control of key genes that code for the proteins involved in in-sulin biosynthesis and secretion and in glucose and incretin actions onbeta-cells (Gosmain et al., 2012). It has been shown that heterozygousmutations in the Pax6 gene can induce glucose intolerance (Yasudaet al., 2002). The loss of Pax6 in the adult islet cells has been demon-strated to affect the expression of multiple target genes involved inthemaintenance of pancreatic endocrine function and glucose handling,resulting in the rapid appearance of diabetic symptoms (Hart et al.,2013). Most recently, it has been shown that Pax6 is involved inCCCTC-binding factor-mediated regulation of beta cell survival (Tsuiet al., 2014). Therefore, the present investigation of the mechanismsbywhich glucose regulates Pax6 in beta-cells should help us understandbeta-cell function and its associated disorders.

In the present study, we show that glucose modulates Pax6 expres-sion through the c-Jun N-terminal kinase (JNK)/p38 mitogen activatedprotein kinase (p38 MAPK) pathways in INS-1E cells. Further, wedemonstrate that tumor-suppressor phosphatasewith tensin homology(PTEN) is one of the possible targets of Pax6 in INS-1E cells.

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Materials and methods

Chemicals

Staurosporine, nifedipine, SP600125, U0126, ionomycin andSB203580 were purchased from Sigma Chemicals Company, SaintLouis,MO, USA. Cell culturemediawere obtained fromHiMedia Labora-tories Pvt. Ltd., Mumbai, India. All of the other chemicals used were ofanalytical grade and were obtained from Sisco Research LaboratoriesPvt. Ltd., Mumbai, India and HiMedia Laboratories Pvt. Ltd., Mumbai,India.

Cell culture

Rat insulinoma-1E (INS-1E) cells were cultured in a humidifiedatmosphere containing 5% CO2 in complete medium, composed ofRPMI-1640 supplemented with 10% heat-inactivated fetal bovineserum, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol, 2 mMglutamine, 10 mM Hepes, 100 units/ml penicillin, and 100 μg/mlstreptomycin. For the glucose regulation experiments, cells weregrown in complete medium and then washed with 3× phosphate-buffered saline before being transferred to low (1 mM) or high(25 mM) glucose-containing media for the indicated times.For inhibitor-based assays, cells were treated with specific inhibitors1 h prior to glucose treatment.

Real time PCR

Briefly, the total RNA from INS-1E cells was isolated according toinstructions from the RNA isolation kit (One step RNA TRIzol Reagent;Biobasic Inc., Markham Ontario, Canada). First-strand cDNA synthesiswas performed using the iScript cDNA synthesis kit (Bio-Rad Laborato-ries, Inc., USA) by following the manufacturer's protocol. The real-timeamplification of the cDNA was achieved using the Brilliant SYBR GreenQPCR Master Mix according to the manufacturer's protocol (AmpliqonA/S; Skovlunde, Denmark). The specific set of primers used for Pax6and β-actin genes were F: 5′-CCAACGACAATATACCCAGTGTGTC-3′ andR: 5′-TGTTGCTGGCAGCCGTCTTGCGTG-3′ and F: 5'-TTCAACACCCCAGCCATGT-3' and R: 5'-TGGTACGACCAGAGGCATACAG-3', respectively.Fold differences in Pax6 expression were calculated using the formula2ΔΔCt.

Western blotting

Cell extracts from INS-1E cells were prepared in lysis buffer (10 mMTris, pH 8.0, 140 mM NaCl, 5 mM MgCl2, 0.2 mM EDTA, 0.5% NonidetP-40, 20% glycerol, 1 mM phenylmethylsulfonyl fluoride, protease andphosphatases inhibitors). Proteins were separated by SDS-PAGE andsubsequently electroblotted onto nitrocellulose membranes. Mem-branes were blocked for 1 h at room temperature in 1× TTBS (20 mMTris, pH 7.4, 150 mM NaCl, 0.1% Triton X-100) supplemented with 5%Carnation nonfat dry milk. After blocking, membranes were incubatedovernight at 4 °C, either with antibodies specific for Pax6, β-actin, cyclinD1 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or PTEN (Cell Signal-ing Technology, USA). Membranes were washed four times for 10 minin TTBS and subsequently incubated for 1 h at room temperature witha secondary horseradish peroxidase-conjugated antibody. Proteinswere visualized using the 3,3'-Diaminobenzidine (DAB) chromogensystem (Bio-Rad Laboratories, Inc., USA). The densitometric analyseswere carried out with lab image platform ver 2.1 software by KapelanBio-Imaging GmbH.

siRNAs transfection

Transfection with small interfering RNAs (siRNAs) was accom-plished using the Eppendorf Multiporator system under optimized

conditions. Silencer negative control (sc-37007) and Pax6 (sc-270113)siRNAswere obtained as annealed oligos fromSanta Cruz Biotechnology,Inc., Santa Cruz, CA. The siRNAswere transfected at a final concentrationof 200 nM.

Assay of cell viability

Cell viability was determined using a colorimetric, 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) assay.Briefly, Pax6 knockdown INS-1E cells were cultured in serum-free low(1 mM) or high (25 mM) glucose-containing media overnight. Next,the MTT solution was added (0.5 mg/ml) to the cells and incubatedfor 4 h. The blue formazan products formed in the INS-1E cells were dis-solved in DMSO and spectrophotometrically measured at a wavelengthof 550 nm.

Data analysis

The values are expressed as means ± standard deviation (SD).Differences between the groups were assessed by one way analysis ofvariance using the GraphPad Prism 6.0 software package for Windows.Post hoc testing was performed for inter-group comparisons usingTukey's multiple comparison test. Values are statistically significant at**P b 0.001 and *P b 0.05.

Results

High glucose suppresses Pax6 expression

In the present study, we investigated whether glucose has any rolein the regulation of Pax6 expression in INS-1E cells. To test this idea,INS-1E cells were incubated on 1 mM (low) or 25 mM (high) glucoseovernight. The mRNA and protein levels were determined by RealTime PCR and Western blotting, respectively. Interestingly, we foundthat the mRNA and protein levels of Pax6 were reduced by 4-foldapproximately in high glucose when compared to low glucose treatedcells (Fig. 1A & B). Further, we have also analyzed the expression levelsof Pax6 in normal glucose (5.5 mM) and osmotic control (25 mMmannitol) media. The protein levels of Pax6 were significantly reducedin high glucose when compared to normal and osmotic controls(Fig. 1C). To test whether high glucose removal restores Pax6 expres-sion, high glucose-treated cellswere switched to low glucose overnight.Interestingly, the removal of high glucose from the culture mediarestored Pax6 protein expression in INS-1E cells (Fig. 1D). These resultsreveal that high glucose suppresses Pax6 expression in INS-1E cells.

High glucose suppression of Pax6 is mediated by phosphorylation

To test whether Pax6 levels are regulated by cellular kinases, wetreated low or high glucose-grown INS-1E beta-cells with the generalprotein kinase inhibitor staurosporine. Staurosporine treatment inhigh glucose-added INS-1E cells significantly increased Pax6 proteinlevels when compared to cells treated with high glucose alone. How-ever, staurosporine treatment in low glucose did not show anychange in Pax6 protein expression when compared to INS-1E cellstreatedwith lowglucose alone (Fig. 2). These results indicate that phos-phorylation event(s) suppress(es) Pax6 expression in high glucose-treated INS-1E beta-cells.

Calcium signaling is not involved in Pax6 regulation

It has been shown that glucose increases intracellular calcium levelsand mediates Ca2+-dependent phosphorylation events in beta-cells(Wollheim and Pozzan, 1984; Alejandro et al., 2010). To identifywheth-er Ca2+-dependent phosphorylation events are involved in the glucoseregulation of Pax6 expression in INS-1E cells, we treated low or high

Fig. 1. High glucose suppresses Pax6 expression in beta-cells. INS-1E cells were cultured in the presence of 1 mM (low glucose) or 25 mM (high glucose) or 5.5 mM (normal glucose) or25 mMmannitol (osmotic control) for overnight and Pax6 gene and protein expressions were analyzed (n= 3). A) Effect of glucose on Pax6 gene expression in INS-1E cells. B) Effect ofglucose on Pax6 protein levels in INS-1E cells. C) Effect of normal glucose and osmotic control on Pax6 protein expression in beta-cells. D) Removal of high glucose restores the expressionof Pax6 in beta-cells. Values represent mean ± S.D. Values are statistically significant at **P b 0.001, *P b 0.05.

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glucose-grown INS-1E beta-cells with the calcium channel blockernifedipine or calcium ionophore ionomycin. However, nifedipine treat-ment, in both low and high glucose-treated cells, showed no alterationsin Pax6 expression when compared to the respective controls (Fig. 3).Similarly, ionomycin treatment also showed no alterations in Pax6expression in INS-1E cells (Supplementary Fig. 1). These data suggestthat calcium signaling is not involved in the regulation of Pax6 expres-sion in INS-1E cells.

JNK/p38 MAP kinase signaling regulates Pax6 expression

Several signal transduction pathways, including JNK, ERK and p38MAPK, are activated by high glucose in pancreatic beta-cells (Evanset al., 2003). To test whetherMAP kinases play a role in the glucose reg-ulation of Pax6 expression, we used specific inhibitors for JNK, p38 andERK/MAP kinases in our study. Interestingly, we found that the JNKinhibitor SP600125 and the p38 MAPK inhibitor SB203580 significantlyincreased Pax6 levels in high glucose-treated INS-1E cells, compared totheir respective controls (Fig. 4A &B). However, ERK inhibition byU0126 did not show any alteration in Pax6 protein expression(Fig. 4C). Overall, these data suggest that JNK/p38MAP kinase activationsuppresses Pax6 expression in high glucose-treated INS-1E cells.

Effect of Pax6 knockdown on PTEN expression and cell viability

PTEN and cyclin D1 are considered critical regulators of cell growthand proliferation. Studies conducted by other investigators in neuronalcells have demonstrated that Pax6 positively regulates PTEN expressionand controls cell growth (Sansom et al., 2009; Zhou et al., 2003). Con-currently, it has also been shown that PTEN is a general negative regula-tor of cyclin D expression (Diao and Chen, 2007). We believe that theglucose regulation of Pax6 may have a role on PTEN and cyclin D1expression in beta cells. To test this idea, we first investigated the effectof glucose on PTEN and cyclin D1 expression in INS-1E cells. Interest-ingly, we found that the protein expression of PTEN is increased andcyclin D1 expression is reduced in low, compared to high, glucose-treated cells (Supplementary Fig. 2). Next, we investigated the effectof glucose on PTEN and cyclin D1 expression in Pax6 knockdown INS-1E cells. Our results demonstrated that the Pax6 knockdown signifi-cantly reduced PTEN expression and concurrently increased cyclinD1 expression in low glucose-treated cells compared to respectivecontrols. Pax6 knockdown also reduced the expression of PTEN inhigh glucose-treated cells; however, this effect was not statisticallysignificant (Fig. 5B). Considering the importance of these transcriptionfactors to cell growth and proliferation, we investigated the effect of

Fig. 2. Staurosporine induces Pax6 protein expression in high glucose. INS-1E cells werecultured in the presence of 1 mM (low glucose) or 1 mM (low glucose) + 100 nM(staurosporine) or 25 mM (high glucose) or 25 mM (high glucose) + 100 nM(staurosporine) for overnight and Pax6 protein expressions were analyzed (n = 3).β-actin levelswereutilized as internal control. Values representmean±S.D. Comparisonsaremade between a—(low glucose versus high glucose) and b—(high glucose versus highglucose + staurosporine). Values are statistically significant at **P b 0.001, *P b 0.05.

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Pax6 knockdown on cell survival in low and high glucose-treated INS-1E cells. Interestingly, we found that the Pax6 knockdown increasedcell viability in both low and high glucose-treated cells and that the

Fig. 3. Calcium signaling is not involved in the regulation of Pax6 in beta-cells. INS-1E cellswere cultured in the presence of 1 mM (low glucose) or 1 mM (low glucose) + 10 μM(nifedipine) or 25 mM (high glucose) or 25 mM (high glucose) + 10 μM (nifedipine)for overnight andPax6protein expressionswere analyzed (n=3).β-actin levelswere uti-lized as internal control. Values represent mean ± S.D. Comparisons are made between a—(lowglucose versushighglucose) andb—(highglucoseversushighglucose+ nifedipine).Values are statistically significant at *P b 0.05, ns—non significant.

effect of Pax6 knockdown on cell viability is more significant in lowglucose-treated cells (Fig. 5C).

Discussion

The paired and homeodomain-containing transcription factor Pax6plays a pivotal role in the development of the endocrine pancreas inaddition to its more commonly known roles in the development of theeye, brain and olfactory system (Cerf, 2006; Osumi et al., 2008; Tsonisand Fuentes, 2006; Nomura et al., 2007). It has been shown that hetero-zygous mutations in the Pax6 gene can induce glucose intolerance(Yasuda et al., 2002). More recently, it has been demonstrated thatPax6 plays roles in the regulation of the insulin 1 and 2, pancreaticduodenal homeobox 1 (Pdx1),masculo neurotic factor A (MafA), glucosetransporter 2 (GLUT2), glucokinase (GK), homeoboxproteinNK-6homo-logue A (Nkx6.1), V-maf musculoaponeurotic fibrosarcoma oncogenehomologue (cMaf) and prohormone convertase 1/3 (PC1/3) genes, allof which are involved in beta-cell function (Gosmain et al., 2012).

Glucose is the principal physiological insulin secretagogue and apotent regulator of beta cell activity and modulates a large number ofgene encoding proteins that are involved in transcription, RNA splicing,cellular maintenance and beta-cell repairs (Meugnier et al., 2007). INS-1E cells display many important characteristics of pancreatic beta-cells,including the expression of GLUT2 and glucokinase, aside from theirhigh insulin content and responsiveness to glucose (Skelin et al.,2010). The high Km of GLUT2 glucose transporters permits rapid glu-cose uptake in these cells, regardless of the extracellular glucose con-centration (Schuit et al., 2001). In the present study, we identifiedthat Pax6 mRNA and protein levels were significantly diminished inhigh glucose compared to low glucose concentrations in INS-1E cells.Here, we have also shown that the kinase inhibitor staurosporineincreases Pax6 expression in high glucose-treated INS-1E beta-cells. Staurosporine is a potent inhibitor of the protein kinases, in-cluding phospholipids/Ca2+-dependent protein kinases and JNK,through its prevention of ATP's binding to the kinase with high affinity(Karaman et al., 2008). Hence, our results indicate that high glucose-activated protein kinase(s) play(s) a role in the suppression of Pax6 inbeta-cells.

Hyperglycemia has been implicated in the activation of many signaltransduction pathways, and it has been demonstrated that high glucoseactivates JNK and p38 MAPK in various cells, including beta-cells(Lanuza-Masdeu et al., 2013;Wilmer et al., 2001). Our data from exper-iments involving specific inhibitors for signal transduction pathwayssuggest that calcium signaling is not involved in the regulation of Pax6expression, but rather, that JNK/p38 MAP kinase activation suppressesPax6 expression at 25mM in glucose treated-INS-1E cells.Most recently,Tsui et al. showed that glucose upregulates theCCCTC-binding factor andconsequently downregulates Pax6 expression through ERK signaling inβ-TC-1-6 cells (Tsui et al., 2014). The findings of their study revealedthat 50 mM and higher concentrations of glucose cause the suppressionof Pax6 expression in β-TC-1-6 cells. The reason for this discrepancy inthe findings can be explained at least in part by the phenotypic varia-tions between these cell types. Rat insulinoma-1E (INS-1E) cells arerelatively more sensitive to glucose due to their higher insulin secretingabilities than mouse pancreatic β-TC-1-6 cells (Skelin et al., 2010; Asfariet al., 1992). Moreover, it has been shown that JNK regulates Pax2 andPax6 expressions in the eye lens and plays a pivotal role in the closureof the optic fissure (Weston et al., 2003). Mikkola et al. have observedthat p38/ERK, not JNK, signaling is involved in the phosphorylation ofthe transactivation domain of Pax6 in zebrafish (Mikkola et al., 1999).Hence, it appears that MAPK pathways function in cell-type andstimulus-dependent manners.

Further, the siRNA-mediated knockdown of Pax6 significantlydecreased the expression of PTENwhile increasing cyclin D1 expressionand cell viability in low glucose-treated INS-1E cells. PTEN is a lipidand protein dual phosphatase and plays a vital role in embryonic

Fig. 4. A. JNK/MAP kinase regulates Pax6 in beta-cells. INS-1E cells were cultured in the presence of 1 mM (low glucose) or 1 mM (low glucose) + 30 μM (SP600125) or 25 mM (highglucose) or 25mM (high glucose) + 30 μM (SP600125) overnight and Pax6 protein expressions were analyzed (n= 3). β-actin levels were utilized as internal control. Values representmean±S.D. Comparisons aremade between a—(lowglucose versushighglucose) and b—(high glucose versushigh glucose+ SP600125). Values are statistically significant at **P b 0.001.B. Inhibition of the p38/MAP kinase induces Pax6 in high glucose. INS-1E cells were cultured in the presence of 1mM (low glucose) or 1mM (low glucose)+10 μM(SB203580) or 25mM(high glucose) or 25 mM (high glucose) + 10 μM (SB203580) overnight and Pax6 protein expressions were analyzed (n = 3). β-actin levels were utilized as internal control. Valuesrepresent mean ± S.D. Comparisons are made between a—(low glucose versus high glucose) and b—(high glucose versus high glucose + SB203580). Values are statistically significantat **P b 0.001 and *P b 0.05. C. Inhibition of the ERK/MAP kinase is not involved in the regulation of Pax6 in beta-cells. INS-1E cells were cultured in the presence of 1 mM (low glucose)or 1 mM (low glucose) + 10 μM (U0126) or 25 mM (high glucose) or 25 mM (high glucose) + 10 μM (U0126) overnight and Pax6 protein expressions were analyzed (n = 3). β-actinlevels were utilized as internal control. Values represent mean ± S.D. Comparisons are made between a—(low glucose versus high glucose) and b—(high glucose versus high glucose +U0126). Values are statistically significant at **P b 0.001 and ns—non significant.

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development, cell growth, apoptosis and cell migration (Diao and Chen,2007). Many previous reports have demonstrated that Pax6 acts as anactivator of PTEN expression and controls cell growth (Sansom et al.,2009; Zhou et al., 2003). Concurrently, it has also been shown thatPTEN is a general negative regulator of cyclin D expression (Diaoand Chen, 2007; Planchon et al., 2008). PTEN induces cell cycle arrestby decreasing the level and nuclear localization of cyclin D1 (Raduet al., 2003). Mahimainathan et al. have shown that high glucosedownregulates PTEN expression in mesangial cells (Mahimainathanet al., 2006). It has also been demonstrated that high glucose stimulatescell proliferation by increasing the expression of cyclin D1 (Okumuraet al., 2002). The results of the present study also well corroborate the

findings of previous studies observing decreased PTEN and increasedcyclin D1 expression in high glucose conditions. From the results of thepresent study, we speculate that high glucose activates the JNK/p38MAPK pathway, thereby downregulating Pax6 expression in INS-1Ecells. The suppression of Pax6 in high glucose, in turn, decreases theexpression of PTEN while increasing cyclin D1. This may be one of thepossible mechanisms subserving the high glucose-induced cell viabilityin beta-cells. However, further experiments are needed to elucidatethe detailed mechanisms by which glucose regulates Pax6 and controlsbeta-cell function.

Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.lfs.2014.06.009.

Fig. 5. Effect of Pax6 knockdown on cell cycle regulators in beta-cells. A) RepresentativeWestern blot to show Pax6 knockdown in INS-1E cells. B) Effect of Pax6 knockdown on PTEN andCyclin D1 in INS-1E cells cultured in the presence of 1 mM (low glucose) or 25 mM (high glucose) overnight. C) Effect of Pax6 knockdown on cell viability in INS-1E cells cultured in thepresence of 1mM(lowglucose) or 25mM(high glucose) overnight (n=3). Values representmean±S.D. Comparisons aremadebetween a—(control siRNA+ lowglucose versus controlsiRNA + high glucose), b—(control siRNA + low glucose versus Pax6 siRNA + low glucose) and c—(control siRNA + high glucose versus Pax6 siRNA+ high glucose). Values are statis-tically significant at **P b 0.001, *P b 0.05 and ns—non significant.

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Conflict of interest statementNIL.

Acknowledgments

The authors gratefully acknowledge the Department of Science andTechnology (No.SR/FT/LS-202/2009) and the University Grants Com-mission (No.40-210/2011-SR), NewDelhi, India for their financial assis-tance. The infrastructure provided by DST-FIST is also gratefullyacknowledged.

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