Drug-DNA Interaction: A Theoretical Study of the Stability of CP-DNA Binding with Thionine

doi:10.4236/ojapps.2012.22013

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Open Journal of Applied Sciences, 2012, 2, 98-103

doi:10.4236/ojapps.2012.22013 Published Online June 2012 (http://www.SciRP.org/journal/ojapps)

Drug-DNA Interaction: A Theoretical Study of the Stability

of CP-DNA Binding with Thionine

Ghazala Yunus*, Seema Srivastava, Vishwambhar Dayal Gupta

Department of Physics, Integral University, Lucknow, India

Email: *[email protected]

Received March 24, 2012; revised April 22, 2012; accepted May 9, 2012

ABSTRACT

The recent study on binding of small molecules to double stranded DNA suggested that the intercalation of a tricyclic

heteroaromatic molecule, thionine, with natural DNA provided thermal stabilization to the complex. In the present

study, we reported theoretical analysis of thionine binding with Clostridium perfringenes DNA duplex (CP-DNA) by

using an amended Zimm and Bragg theory, to explain the melting behaviour and heat capacity of CP-DNA with and

without thionine binding. The experimental models of Paul et al. (2010) have been used for the study. The sharpness of

transition has been examined in terms of half width and sensitivity parameter (∆H/σ). The results of theoretical ap-

proach suggested that the various parameters such as transition profile, sharpness of the transition, heat capacity curve

and half widths are in good agreement with the experimental measurements for binding of thionine. Therefore, the pro-

posed theoretical analysis may be useful in order to understand interaction of small molecules to DNA that may be ap-

plied in the process of drug development and for designing more potential DNA binding therapeutic molecules.

Keywords: Thionine; DNA Binding; Transition Profile; Heat Capacity; Drug Design

1. Introduction

The study of the interaction of small molecules with

DNA is a field of high tropical interest and the number

and verity of techniques devoted to evidence drug-DNA

interactions is continuously growing. The binding of het-

erocyclic aromatic molecules to nucleic acids has re-

ceived considerable attention over the past several years

due to their relevance in various biological applications

including cancer chemotherapy [1-8]. Accordingly, a

number of experimental studies are conducted to under-

stand the nature and thermodynamics of heterocyclic

aromatic molecules and nucleic acid interaction [7,9-16].

Thionine (3,7-Diamino-5-phenothiazinium) (Figure 1), a

tricyclic heteroaromatic molecule, has been studied for

its intercalative interaction with duplex DNA [13] and

photoinduced mutagenic actions on binding to DNA [11].

On the basis of satellite hole spectroscopy study Chang et

al. [17] recommended that thionine binds specifically

with guanine-cytosine (GC) contents of duplex DNA.

However, Tuite and Kelly [13] suggested that (GC)

specificity of thionine binding was not very marked. The

model proposed by Hecht et al. [18,19] suggested that

thionine exists in two different tautomeric forms and the

amino form is intercalated while the imino form is not.

Although these data provide somewhat cont radicting

data on the GC specificity of thionine binding to linear

double stranded DNA. A recent study [18] by pressure

tuning hole burning spectroscopy has conclusively

shown an external stacking mode of binding of thionine

to quadruplex structures. Thus, although some structural

data is available, the same is not conclusive and the

thermodynamics of thionine binding to DNA is not yet

elucidated. The calorimetric analysis of thionine binding

to a DNA duplex was reported by Paul et al. [7]. They

also investigated the structural and thermodynamic as-

pects of thionine binding to natural DNAs of varying

base composition. They showed strong binding of thion-

ine with CP-DNA which increases the thermal stability

of the duplex, and at saturation the duplex melts with a

Tm some 6.3˚C above that of the free duplex [7]. They

also concluded that binding of thionine to CP-DNA is an

exothermic process and there is involvement of multi-

plicity of non covalent interactions in the binding process.

In the present study, we have attempted to understand the

effect of thionine binding on a native DNA duplex using

the model of Paul et al. [7] who studied the thermal and

*Corresponding author. Figure 1. Chemical structure of thionine.

G. YUNUS ET AL. 99

thermodynamic behaviour of thionine binding to CP-DNA.

To elucidate lambda point anomalies in heat capacities

and order-disorder transition in thionine bounded and

unbounded DNA duplexes, we used amended Zimm and

Bragg theory which is considered initially for the helix

coil transitions in polypeptides [20-22]. The effect of

thionine binding is reflected in the change in nuclea-

tion parameter, which is an inverse measure of binding

strength.

2. Theory

The results of the structural studies proposed by Paul et

al. [7] for the complex of heterocyclic aromatic mole-

cules with DNA on the basis of spectroscopic and calo-

rimetric study, as mentioned above, suggested that thio-

nine binds strongly with the Clostridium perfringenes

DNA that resulted in thermal stabilization of the complex.

The binding of thionine to the AT rich Clostridium per-

fringenes DNA was intercalative and favored by higher

entropic contribution indicating the stronger perturbation

of the water structure associated with the AT sequences

[7]. However, the system remains a highly co-operative

one therefore the co-operative transition theory could be

applied to explain the melting profile and temperature

dependence of thermodynamical parameter, such as heat

capacity. Therefore, we can use amended Zimm and

Bragg theory [20] which is described in our previous

publication [23]. Briefly, the above mentioned theory

consists of writing an Ising matrix for a two-phase sys-

tem, the bounded state and unbounded state. As dis-

cussed earlier [21,22,24-26] and by Zimm and Bragg

[20], the Ising matrix M can be written as:

12 1212 12

rkh

rr rr k

kr kk h

hh rh h

fff

ff ff f

ff ff

M (1)

where fr, fh and fk are corresponding base pair partition

functions’ contributions in the three states i.e. ordered,

disordered and boundary or nucleation. The eigen values

of M are given by:









rh rhrk

ff ffff

























(2)

Since we are dealing with a finite system hence the

effect of initial and final states becomes important. The

contribution of the first segment to the partition function

is given by:





,0,0

fU (3)

where the column vector V gives the state of the last

segment,

V (4)

The partition function for an N-segment chain is given

by;

1N

ZUMV (5)

The matrix T which diagnolizes M consists of the

column vectors given by:





MXX (6)

where,

X.

By substituting the values of M from Equation (6), we

get:



1112 12

12 1212 12

1/21 2

1 1 1

()

rk rk

hr hr

ff ff

ff ff ff













T (7)

Similarly, we get 1



T from the matrix equation



YMY (8)

where, 1, 2, 3

YYY











Again by substituting the values of M from Equation

(1) in Equation (8), we get:















12 1212 12

111

12 1212 12

222

12 1212 12

333

rk krh

cf fcff f









T (9)

The normalization constants are:

12 21

CC C



 







 (10)

If we let 1



TMT be the diagonalized form of M,

the partition function can be written as:

N

ZUT TV (11)

G. YUNUS ET AL.

100

On substituting the values from Equations (1), (3), (4),

(7), (9) and (10) in Equation (11), the partition function

becomes:

here s is propagation parameter, which for simplicity is

assumed to be 1. In fact, in most of the systems, it is

found to be close to unity. If Ar and Ah are the absorbance

in disordered and ordered states, respectively, the total

absorption can be written as:

11 21



Z (12)

The fraction of the segments in the disordered form is

given by





rrr h

QAQ A (14)

The extension of this formalism to specific heat (Cv) is

straight forward. The specific heat is related to the molar

enthalpy and entropy changes in the transition from state

I to II. From the well known thermodynamic relations,

free energy and internal energy are ln

KT Z and









2,UT TFT



 respectively. Differentiating in-

ternal energy with respect to temperature we get the spe-

cific heat:





ln ln

QZf



N

Solving the above equation, we get:















1211

121

22 2

AP s

QPPN





  (13)

where 12











vkm

CUTNHRTSQS











(15)



rh kr

Aff ff













where



is the molar change in enthalpy about the

transition point, S is entropy which is equal to









exp11 m

SHRTT





 



,

T is the transition temperature, and

 











 









2112 1

121

11211211

rPSASA P

QPA

SS S

PSA PSA PS S





 

















 

 



 



  

 

 



 

 

 



 

 





with



SNS













 



 



 



 



























bound and free thionine [7]. When tricyclic heteroaro-

matic thionine binds with natural Clostridium perfringe-

nes DNA duplex, the structure of DNA duplex still re-

mains a very much co-operative and thus two-state the-

ory of order-disorder transition is applicable. The Zimm

and Bragg theory [7] is amended so as to consider or-

dered (bounded/unbounded) and disordered states as the

two states which co-exist at the transition point. The

transition is characterized mainly by the nucleation pa-

rameter and overall change of enthalpy/entropy, which

are also the main thermodynamic forces driving the tran-

sition. The change in enthalpy obtained from differential

scanning calorimetric measurements takes all this into

account. This is evident from the enthalpy change and

changes in other transition parameters, such as nucleation

parameter (σ) and melting point (Table 1). The melting

temperature of the duplex was increased after binding

with thionine at saturation. At saturation, the duplex

melts with a Tm some 6.5˚C above that of the free duplex.

The sharpness of the transition can be looked at in terms

of half width and a sensitivity parameter defined as

(∆H/σ). The variation of these parameters systematically

reflects that the transition is sharp in case of unbounded

state and goes blunt with thionine saturation. In case of



1PS SP



 and kr



; σ is the nucleation

parameter and is a measure of the energy expanded/re-

leased in the formation (uncoiling) of first turn of the

ordered/disordered state. It is related to the uninterrupted

sequence lengths [20]. The volume heat capacity Cv has

been converted into constant pressure heat capacity Cp by

using the Nernst-Lindemann approximation [26]:





CC RACTCT vm

(16)

where A0 is a constant often of a universal value [3.9 ×

10–9 (Kmol)/J–1] and Tm is the melting temperature.

3. Results

3.1. Transition Profiles

The binding affinity of thionine with C-DNA has been

studied by spectroscopic, fluorescence and dialysis meth-

ods which showed strong intercalative binding and en-

abled the assumption of two state systems consisting of

G. YUNUS ET AL. 101

λ-transition, the same trend in the sharpness of transition

is seen between the thionine bounded as well as un-

bounded curves. As expected, the sharpness is better in

unbounded, as compared to bounded state. The various

parameters, which give transition profiles in best agree-

ment with the experimental measurements for binding of

thionine to CP-DNA, are given in Table 1.

The transition profiles and heat capacity for un-

bounded DNA duplex and bounded with thionine are

shown in Figure 2. Minor insignificant deviation at the

tail ends is primarily due to the presence of various dis-

ordered states and presence of short helical segments

found in the random coil states. Figure 2 A shows ex-

perimental and calculated transition curves for the duplex

in the absence of thionine and B shows the transition

when the duplex is saturated with thionine (thionine/

DNA = 0.4). As expected, a cooperative transition profile

is observed with calculated data. The conclusion deduced

from the theoretical data is consistent with the directly

measured binding enthalpy by Paul et al. [7] determined

through DSC.

Table 1. Transition parameters for thionine binding to CP-

DNA.

Parameters Unbounded

CP-DNA

CP-DNA saturated

with thionine

Tm (K) 345 351.5

∆H Kcal/M bp 3.06 × 103 3.24 × 103

σ 3 × 10–4 5 × 10–4

N 98 90

Ah 0 0

Ar 1 1

Half width (Exp.) 5 6.5

Half width (Theo.) 5 6.5

Sensitivity parameter (∆H/σ) 102 × 105 648 × 104

Figure 2. Heat capacity and transition profiles (inset) for

thionine bounded and unbounded CP-DNA. A: Unbounded

state; B: Bounded state at saturation. [(—) calculated and

(••••) experimental values].

3.2. Heat Capacity

The conformational and dynamical states of a macromo-

lecular system are characterized by heat capacity which

is second derivative of the free energy and has been cal-

culated by using Equation (14). These heat capacity

curves with λ-point anomaly are shown in Figure 2 along

with their transition profile. The theoretically obtained

heat capacity profiles agreed with the experimentally

reported ones and could be brought almost into coinci-

dence with the use of scaling factors, which is very close

to one in transition profiles and slightly higher for the

heat capacity curves. The sharpness of the transition can

be characterized by the half widths of the heat capacity

curves that are in good agreement in both experimental

and theoretical graphs.

4. Discussion

The present study concluded that the natural CP-DNA

molecule is an extremely co-operative structure and when

thionine bind to it the co-operativity is not so much dis-

turbed. Therefore, the amended Zimm and Bragg theory

(phase transitions theory) can be effectively applied to it.

It generates the experimental transition profile and λ-

point heat capacity anomaly successfully. These results

will allow us to assess the thermodynamic profile of the

binding process. Our theoretical studies of heterocyclic

aromatic molecules binding are being extended to other

synthetic and natural nucleic acids. The theoretical data

obtained also demonstrated that the binding of thionine

to CP-DN is an endothermic process and that this binding

increases the melting temperature of the duplex as sup-

ported by calorimetric measurement. The specific bind-

ing and intercalation of thionine with DNA have been

studied by using spectroscopic methods [13,17]. How-

ever, Paul and her coworkers used optical melting and

DSC techniques to understand the interaction of small

molecule, thionine, with double stranded deoxyribonu-

cleic acid, native CP-DNA [7]. The binding of thionine

stabilized CP-DNA and ΔTm value of about 6.3˚C was

obtained under saturating condition. Accordingly, we can

interpret our theoretical results in the context of the spe-

cific structural features of the complex as deduced from

their spectroscopic/DSC data. Inspection of Figures 2 A

and B reveals that the transition of the thionine-saturated

CP-DNA duplex is significantly broader than the transi-

tion of the thionine-free duplex. Thus, in addition to af-

fecting the transition enthalpy and the melting tempera-

ture, binding of heterocyclic aromatic molecules also

alters the nature of the transition as reflected by the in-

crease in transition width in experimental and calculated

(theoretical) both data. In recent years, an increased un-

derstanding of the role played by nucleic acids in bio-

logical systems made DNA an alternative candidate for

G. YUNUS ET AL.

102

the development of new drugs. The successful applica-

tions of molecular modeling in virtual ligand screening

and structure-based design of organic and inorganic mo-

lecules that target DNA are highlighted by Ma et al. [27].

The interactions of drugs and calf thymus DNA were

also investigated by using non-linear fit analysis [28].

Other recent literatures also reviewed that the study of

DNA interaction with a small molecules is of high inter-

est [7,29-32]. Thus, the present theoretical analysis can

be applied to understand bimolecular interaction and may

also be implicated to the process of drug development in

biomedical industries.

5. Acknowledgements

Technical assistance from Dr. Mohammed Kuddus, De-

partment of Biotechnology, Integral University, Lucknow

is gratefully acknowledged.

REFERENCES

[1] M. J. Waring, “DNA Modification and Cancer,” Annual

Reviews Biochemistry, Vol. 50, 1981, pp. 159-192.

doi:10.1146/annurev.bi.50.070181.001111

[2] L. H. Hurley, “Secondary DNA Structures as Molecular

Targets for Cancer Therapeutics,” Biochemical Society

Transactions, Vol. 29, No. 6, 2001, pp. 692-696.

[3] L. H. Hurley, “DNA and Its Associated Processes as Tar-

gets for Cancer Therapy,” Nature Reviews Cancer, Vol. 2,

No. 3, 2002, pp. 188-200. doi:10.1038/nrc749

[4] R. Martinez and L. Chacon-Garcia, “The Search of DNA-

Intercalators as Antitumoral Drugs: What It Worked and

What Did Not Work,” Current Medicinal Chemistry, Vol.

12, No. 2, 2005, pp. 127-151.

[5] R. Palchaudhuri and P. J. Hergenrother, “DNA as a Tar-

get for Anticancer Compounds: Methods to Determine

the Mode of Binding and the Mechanism of Action,”

Current Opinion in Biotechnololy, Vol. 18, No. 6, 2007,

pp. 497-503. doi:10.1016/j.copbio.2007.09.006

[6] M. Maiti and G. S. Kumar, “Molecular Aspects on the

Interaction of Protoberberine, Benzophenanthridine, and

Aristolochia Group of Alkaloids with Nucleic Acid Struc-

tures and Biological Perspectives,” Medicinal Research

Reviews, Vol. 27, No. 5, 2007, pp. 649-695.

doi:10.1002/med.20087

[7] P. Paul, M. Hossain, R. C. Yadav and G. S. Kumar, “Bio-

physical Studies on the Base Specificity and Energetics of

the DNA Interaction of Photoactive Dye Thionine: Spec-

troscopic and Calorimetric Approach,” Biophysical Chem-

istry, Vol. 148, No. 1-3, 2010, pp. 93-103.

doi:10.1016/j.bpc.2010.02.015

[8] K. Bhadra and G. S. Kumar, “Therapeutic Potential of

Nucleic Acid-Binding Isoquinoline Alkaloids: Binding

Aspects and Implications for Drug Design,” Medicinal

Research Reviews, Vol. 31, No. 6, 2011, pp. 821-862.

doi:10.1002/med.20202

[9] J. Y. Du, X. H. Huang, F. Xu, Y. Y. Feng, W. Xing and T.

H. Lu, “Spectral Study on the Interaction Mechanism

between Thionine and Calf Thymus DNA,” Guang Pu

Xue Yu Guang Pu Fen Xi, Vol. 25, No. 9, 2005, 1435-

1448.

[10] S. Jockusch, D. Lee, N. J. Turro and E. F. Leonard, “Photo-

Induced Inactivation of Viruses: Adsorption of Methylene

Blue, Thionine, and Thiopyronine on Beta Bacteriophage,”

Proceeding of National Academy of Sciences USA, Vol.

93, No. 15, 1996, pp. 7446-7451.

|doi:10.1073/pnas.93.15.7446

[11] C. Dohno, E. D. A. Stemp and J. K. Barton, “Fast Back

Electron Transfer Prevents Guanine Damage by Photoex-

cited Thionine Bound to DNA” Journal of American

Chemical Society, Vol. 125, No. 32, 2003, pp. 9586-9587.

doi:10.1021/ja036397z

[12] Y. Xu, L. Yang, X. Ye, P. He and Y. Fang, “Impedance-

Based DNA Biosensor Employing Molecular Beacon

DNA as Probe and Thionine as Charge Neutralizer,”

Electroanalysis, Vol. 18, No. 9, 2006, pp. 873-881.

doi:10.1002/elan.200503465

[13] E. M. Tuite and J. M. Kelly, “The Interaction of Methyl-

ene Blue, Azure B, and Thionine with DNA: Formation

of Complexes with Polynucleotides and Mononucleotides

as Model Systems,” Biopolymers, Vol. 35, No. 5, 1995,

pp. 419-433. doi:10.1002/bip.360350502

[14] Y. J. Ihaya and T. Nakamura, “Optical Activity of Nu-

cleic Acid-Thionine Complexes,” Bulletin of the Chemi-

cal Society of Japan, Vol. 44, 1971, pp. 951-957.

[15] C. V. Kumar and E. H. Asuncion, “DNA Binding Studies

and Site Selective Fluorescence Sensitization of an An-

thryl Probe,” Journal of American Chemical Society, Vol.

115, No. 19, 1993, pp. 8547-8553.

doi:10.1021/ja00072a004

[16] E. M. Tuite and J. M. Kelly, “New Trends in Photobiol-

ogy: Photochemical Interactions of Methylene Blue and

Analogues with DNA and Other Biological Substrates,”

Journal of Photo chemistry and Photobiology, B: Biology,

Vol. 21, No. 2-3, 1993, pp. 103-124.

doi:10.1016/1011-1344(93)80173-7

[17] T. C. Chang, Y. P. Yang, K. H. Huang, C. C. Chang and

C. Hecht, “Investigation of Thionine-DNA Interaction by

Satellite Hole Spectroscopy,” Optics and Spectroscopy,

Vol. 98, No. 5, 2005, pp. 716-721.

doi:10.1134/1.1929049

[18] C. Hecht, J. Friedrich and T. C. Chang, “Interactions of

Thionin with DNA Strands: Intercalation versus External

Stacking,” Journal of Physical Chemistry B, Vol. 108, No.

29, 2004, pp. 10241-10244. doi:10.1021/jp0372993

[19] C. Hecht, P. Hermann, J. Friedrich and T. C. Chang,

“Thionin in a Cyclodextrin Nanocavity: Measuring Local

Compressibilities by Pressure Tuning Hole Burning Spec-

troscopy,” Chemical Physics Letters, Vol. 413, No. 4-6,

2005, pp. 335-341. doi:10.1016/j.cplett.2005.08.002

[20] B. H. Zimm and J. K. Bragg, “Theory of the Phase Tran-

sition between Helix and Random Coil in Polypeptide

Chains,” Journal of Chemical Physics, Vol. 31, No. 2,

1959, pp. 526-535. doi:10.1063/1.1730390

[21] S. Srivastava, V. D. Gupta, P. Tandon, S. Singh and S. B.

Katti, “Drug Binding and Order-Order and Order-Disor-

G. YUNUS ET AL.

103

der Transitions in DNA Triple Helices,” Journal of Mac-

romolecular Science Physics, Vol. 38, No. 4, 1999, pp.

349-366. doi:10.1080/00222349908212437

[22] S. Srivastava, S. Srivastava, S. Singh and V. D. Gupta,

“Stability and Transition in a DNA Tetraplex: A Model

for Telomeres,” Journal of Macromolecular Science Phy-

sics, Vol. 40, No. 1, 2001, pp. 1-14.

doi:10.1081/MB-100000050

[23] G. Yunus, S. Srivastava and V. D. Gupta, “Stability of

DNA Binding with Dipyrandium: A Theoretical Study,”

International Journal of Physical Sciences, Vol. 6, No. 36,

2011, pp. 8151-8156. doi: 10.5897/IJPS11.1335

[24] S. Srivastava, I. A. Khan, S. Srivastava and V. D. Gupta,

“A Theoretical Study of the Stability of DNA Binding

with Cis/Trans Platin,” Indian Journal of Biochemistry

and Biophysics, Vol. 41, No. 6, 2004, pp. 305-310.

[25] N. Poklar, D. S. Pilch, S. J. Lippard, E. A. Redding, S. U.

Dunham and K. J. Breslauer, “Influence of Cisplatin In-

trastrand Crosslinking on the Conformation, Thermal Sta-

bility, and Energetics of a 20-mer DNA Duplex,” Proceed-

ing of National Academy of Sciences USA, Vol. 93, No.

15, 1996, pp. 7606-7611. doi:10.1073/pnas.93.15.7606

[26] K. A. Roles and B. Wunderlich, “Heat Capacities of Solid

Poly(Amino Acids). I. Polyglycine, Poly(L-Alanine), and

Poly(L-Valine),” Biopolymers, Vol. 31, No. 5, 1991, pp.

477-487. doi:10.1002/bip.360310503

[27] D. L. Ma, D. S. Chan, P. Lee, M. H. Kwan and C. H.

Leung, “Molecular Modeling of Drug-DNA Interactions:

Virtual Screening to Structure-Based Design,” Biochimie,

Vol. 93, No. 8, 2011, pp. 1252-1266.

doi:10.1016/j.biochi.2011.04.002

[28] J. Yuan, W. Guo and E. Wang, “Oligonucleotide Stabi-

lized Silver Nanoclusters as Fluorescence Probe for Drug-

DNA Interaction Investigation,” Analitica Chimica Acta,

Vol. 706, No. 2, 2011, pp. 338-342.

doi:10.1016/j.aca.2011.08.043

[29] F. Araya, G. Huchet, I. McGroarty, G. G. Skellern and R.

D. Waigh, “Capillary Electrophoresis for Studying Drug-

DNA Interactions,” Methods, Vol. 42, No. 2, 2007, pp.

141-149. doi:10.1016/j.ymeth.2006.09.006

[30] M. M. Islam, S. R. Chowdhury and G. S. Kumar, “Spec-

troscopic and Calorimetric Studies on the Binding of Al-

kaloids Berberine, Palmatine and Coralyne to Double

Stranded RNA Polynucleotides,” Journal of Physical

Chemistry B, Vol. 113, No. 4, 2009, pp. 1210-1224.

doi:10.1021/jp806597w

[31] V. Gonzalez-Ruiz, A. I. Olives, M. A. Martin, P. Ribelles,

M. T. Ramos and J. C. Menendez, “An Overview of Ana-

lytical Techniques Employed to Evidence Drug-DNA In-

teractions. Applications to the Design of Genosensors,” In:

M. A. Komorowska and S. Olsztynska-Janus, Eds., Bio-

medical Engineering, Trends, Research and Technologies,

2011, InTech, Castle Rock, pp. 65-90. doi:10.5772/13586

[32] A. Kunwar, S. Emmanuel, U. Singh, R. K. Chittela, D.

Sharma, S. K. Sandur and I. K. Priyadarsini, “Interaction

of a Curcumin Analogue Dimethoxycurcumin with DNA,”

Chemical Biology and Drug Design, Vol. 77, No. 4, 2011,

pp. 281-287. doi:10.1111/j.1747-0285.2011.01083.x