DOI: 10.1002/cbic.201200193
Resveratrol and Health: The Starting Point
Lucia Biasutto,[a] Andrea Mattarei,[b] and Mario Zoratti*[a]
Juan Ponce de Leon’s 16th century search for the fountain of
youth has been often used as a metaphor for research on resveratrol, the most famous of polyphenols, purported to have
nearly miraculous virtues. Like the Spaniard’s trek through wild
territories, the investigation of resveratrol’s effects and of their
underlying mechanisms has proven long and dogged by setbacks. De Leon’s search eventually proved fruitless; the fountain of youth was but a figment of human wishful thinking. Is
resveratrol, instead, for real? Clinical trials, still inadequate,
have so far neither confirmed nor negated that it can have an
impact on human health.[1] Any number of studies, on the
other hand, have shown that the biochemistry is there, at least
potentially. As usual, fairly intricate biochemistry is involved,
which the paper recently published in Cell by Sung-Jun Park,
Jay H. Chung and co-workers[2] valiantly tries to elucidate.
The framework is the relationship between calorie restriction
(CR), resveratrol (1) and Sirt1.[3] CR, that is, limiting food intake,
can prolong life. This is a phenomenon that is complex (also
experimentally) and still only incompletely understood; its
mechanistic features can depend on details such as whether
CR is life-long or begins after maturity.[4] The major metabolism-regulating pathways, which involve insulin/IGF-1, mTOR/
S6K and AMPK, are implicated. Sirt1, the “founding member”
of a family of seven NAD-dependent deacetylases, has been
proposed as a key player.[5] Among other actions, Sirt1 can deacetylate transcription factors, for example, FOXO, thereby
modulating their activity. Gain-of-function studies indicate that
Sirt1 can act as a positive regulator of telomere length and of
homologous recombination.[6]
A connection between resveratrol and Sirt1 emerged when
resveratrol was reported to induce life extension in yeast; the
effect was attributed to a direct activating effect on Sir2 (the
[a] Dr. L. Biasutto, Dr. M. Zoratti
CNR Institute of Neuroscience and Department of Biomedical Sciences
University of Padova
Viale Giuseppe Colombo 3, 35131 Padova (Italy)
E-mail: [email protected]
[b] Dr. A. Mattarei
CNR Institute of Neuroscience and Department of Chemical Sciences
University of Padova
Via Marzolo 1, 35131 Padova (Italy)
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yeast orthologue of Sirt1).[7] This was exciting and seemed to
make sense. Resveratrol was observed to increase the response
in fluorescence assays measuring Sirt activity on the acetylated
tetrapeptide “fluor-de-lys”. However subsequent reports (e.g.,
ref. [8]) indicated that the Sirt-activating effect is substrate dependent, that is, it is only observed when the fluor-de-lys peptide is used as a substrate for the enzyme in in vitro assays. A
case of bad luck. Nonetheless, the evidence still pointed to
Sirt1 activation in cells exposed to resveratrol (and other polyphenols). An indirect mechanism had to be operating, and in
fact had already been partly delineated before the work of
Park and colleagues.
Sirt1 activity is upregulated by an increase in NAD + .[9] NAD +
can increase downstream of the activation of AMPK, a key
component of an energy level-sensing feedback system that
controls metabolic homeostasis.[9a, 10] This Ser/Thr kinase is activated by AMP and inhibited by ATP. Thus it responds to the
AMP/ATP ratio, which is a sensitive measure of the metabolic
status of the cell. Nucleotide binding also affects phosphorylation and consequent activation by the upstream kinases LKB1
and CaMKKb. The enzyme can also be activated, through LKB1
and independently of the AMP/ATP ratio, by reactive oxygen
species (ROS).[11] One of its tasks is to stimulate metabolic
responses and mitochondrial biogenesis in order to prevent
energy depletion when ATP levels drop (hypoxia, nutrient starvation, increased consumption). This results in an increase in
the cellular NAD + /NADH ratio because of the upregulation of
fatty acid oxidation.[12] Activation of AMPK by resveratrol (and
other phytochemicals) is clearly supported by data from several labs (e.g., ref. [13]) This action is Sirt1-independent; conversely, a functional AMPK is necessary for Sirt1 activation by
resveratrol.[13c, 14] Resveratrol has been found to increase the
NAD + /NADH ratio in an AMPK-dependent manner.[13c] Thus,
resveratrol-induced Sirt1 activation is considered to involve
AMPK.
Age-related metabolic disorders, not longevity, are addressed by Park and colleagues. AMPK has a large role in glucose-stimulated insulin secretion, so much so that a major
approach to treatment for type 2 diabetes currently relies on
AMPK activators such as metformin. Even though the mechanisms involved are not completely known, Sirt1 might be involved.[15] AMPK is important for the metabolic syndrome also
because of its role as a nutrient sensor in the hypothalamus,
a brain region critical for energy homeostasis and the control
of feeding behaviour. Indeed, resveratrol offers promise as an
anti-obesity and anti-diabetic agent.[16]
But how resveratrol brings about AMPK activation remained
uncertain. The effect has been reported to depend on
LKB1.[13a, 17] Vingtdeux et al.[13b] have obtained data pointing to
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a resveratrol-induced increase in cytosolic Ca2 + levels and activation of CaMKKb, the other recognised kinase upstream of
AMPK. Another hypothesis[9] relies on the concept of hormesis,
and proposes that the effect is actually a consequence of mild
inhibition of mitochondrial ATP production by resveratrol,
which has been reported to inhibit the F0F1 ATPase (IC50 :
19 mm)[18] as well as the respiratory chain.[18b]
Thus the primum movens remained uncertain. Now Park
et al.[2] have provided powerful support for one of these mechanistic schemes, the one identified by Vingtdeux et al.,[13b] and
have gone several steps further, reaching the spring (or at
least one of the springs), of the metaphoric fountain. The signalling cascade (Figure 1) begins with a direct inhibition of
phosphodiesterases by resveratrol. The authors tested its activity on recombinant PDE-1 to -5, observing a competitive inhibition for -1, -3 (active on both cGMP and cAMP) and -4 (cAMP).
PDE4 is considered to be the most important diesterase for
the modulation of cAMP levels. Accordingly, cAMP was found
to increase by up to a factor of 1.5–1.8 in C2C12 myotubes
treated with resveratrol. The effect was close to maximum at
20 mm resveratrol and can be transient, declining after about
one hour. Given the low bioavailability of resveratrol (see
below), concentrations in the several-mm range are unlikely to
be reached in vivo.
Through a well-delineated cascade of events (Figure 1), this
cAMP increase leads to the release of Ca2 + from the reticulum
and activation of CamKKb, upstream of AMPK. The possibility
of a concurrent involvement of LKB1 was not investigated, perhaps because resveratrol-induced increases in the phosphorylation levels of AMPK took place also in the presence of siRNA
against the catalytic subunit of PKA, an activating kinase of
LKB1.[19] An increase in cAMP levels would, however, be expected to determine an increase in PKA, and thus LKB1, activity
(barring compartmentalisation effects). LKB1 is also thought to
mediate AMPK activation by ROS.[11a] Park et al. consider resveratrol to be an anti-oxidant, but a direct verification was not
performed, and the evidence from a previous publication[13c] is
questionable. Several reports have shown that, at concentrations in the range used by Park et al., resveratrol can induce
ROS production in cultured cells, so that the hypothesis that
a pro-oxidant effect might contribute to the activation of
AMPK remains. In fact, Park et al. report that, at 300 mm resveratrol, AMPK phosphorylation is no longer affected by the inhibition of CamKKb. An involvement of LKB1 thus cannot be discounted, and is in fact considered possible by the authors
themselves.
Although contribution by this pathway remains a possibility,
the competitive inhibition of PDEs is now brought to the fore.
The “competitive” is important here. Concentrations of resveratrol of up to 50 mm were used in these experiments. Lineweaver–Burk plots of cAMP hydrolysis are shown for PDE3. By assuming fully competitive inhibition one can roughly estimate
a Km for cAMP of about 0.5 mm, and a Ki of about 50 mm (by
using abscissa intercepts of 1.8 and 0.9 mm 1 for no resveratrol and 50 mm resveratrol, respectively). A less drastic difference is indicated by a competition study in which resveratrol
was pitted against cAMP as the inhibitor of binding of a fluoChemBioChem 2012, 13, 1256 – 1259
Figure 1. A cartoon showing the signalling cascade identified by Park and
colleagues. While caloric restriction brings about an increase in cAMP levels
by stimulating adenylate cyclase, resveratrol and Rolipram do the same by
inhibiting PDE4 and (resveratrol) other phosphodiesterases. cAMP elevation,
which would be expected also to increase PKA activity, activates Epac1,
a GEF of Rap-1 and -2, small GTPases of the Ras family. A specific activator
of Epac1 has much the same downstream effects as resveratrol. The steps
immediately below Epac1 are proposed to involve PLCe (activated by
RapGTP), CamKII-mediated phosphorylation of the ryanodine receptor and
perhaps other activities. In fact, a PLC inhibitor prevents downstream
events. Ryanodine-sensitive release of Ca2 + from the sarcoplasmic reticulum
appears to be important. The IP3 receptor is not considered to be involved
because a specific inhibitor is without effect, but its expression level in
C2C12 myoblasts, the cells used, was not assayed. Ca2 + chelators block resveratrol’s effects. Ca2 + activates CamKKb, one of the upstream kinases of
AMPK, the master kinase believed to orchestrate the effects of resveratrol
on metabolism and mitochondrial function. The possible involvement of
LKB1, the other AMPKK, in the upregulation of AMPK is also shown.
rescent cAMP analogue to recombinant PDE3. In any case, the
IC50 for inhibition of PDE activity is found to range from about
6 mm for PDE1 to about 14 mm for PDE4. Thus, the affinity of
PDEs for resveratrol would appear to be lower, plausibly one
order of magnitude lower, than that for cAMP. cAMP in cells is
compartmentalised, and its concentration varies depending on
conditions, but it can be taken to be roughly in the 1 mm
range; Park et al. measured 0.4–0.6 pmol per mg protein in
skeletal muscle and white adipose tissue, in agreement with
such an estimate. Thus, for an effective competition, one
would presumably need resveratrol concentrations in the
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M. Zoratti et al.
order of at least a few mm units. The titration with resveratrol
of cellular cAMP in cultured myotubes by Park et al. is in agreement with this notion.
But now the ever-present problem of bioavailability and metabolism surfaces. Resveratrol, with its phenolic hydroxyls, is a
ready-made substrate for phase II metabolism by the glucuronyl- and sulfo-transferases of enterocytes and of the liver. In
pharmacokinetics studies, free resveratrol is found only transiently in blood and then at very low concentrations.[20] Most in
vitro studies—employing concentrations in the tens-of-mm
range—do not really reflect in vivo situations. Of course, this is
a problem no matter what the mechanism of action of resveratrol. In the hands of Park and co-workers, administration to
mice of a high single dose of resveratrol (100 mg per kg by
gavage, as mentioned in Figure S1 A, or 20 mg per kg i.p., as
mentioned in the Experimental Procedures) resulted in an increase in the amount of cAMP from ~ 0.4 to ~ 0.5 nmol per g in
skeletal muscle, and from ~ 0.6 to ~ 0.75 nmol per g in white
adipose tissue. The difference was significant at 45 min after
administration, but, at least in muscle, it had largely disappeared at 60 min. In other experiments, which led to mitochondrial DNA increasing by some 40 %, the mice were given
400 mg per kg per day (equivalent to some 30 g per day for
an average human) for 14 weeks. Thus, the data reviewed here
might be of little physiological relevance for humans, even
those who consume a resveratrol-enriched diet. How can one
reconcile the reports of dietary resveratrol effectiveness and
the low levels in circulation? Perhaps the relevant pharmacokinetic compartment is not blood, but tissue. Data on the
amounts and nature of resveratrol and its metabolites in
organs are few, but there is evidence that a considerable fraction of the resveratrol in tissues could be present as such, in
amounts ranging up to 1 nmol per g.[21] This makes sense, as
resveratrol is less hydrophilic than its conjugation metabolites.
Again, these results were obtained after acute administration
of large doses. Regeneration of the aglycone from its glucuronide might also be important.[1b] The matter certainly deserves
further attention, as do attempts to make resveratrol more bioavailable.[22]
Regardless, the work by Park et al. is notable because it outlines a signalling cascade that is initiated by inhibition of PDE4
and has important metabolic effects. Many experiments actually utilise Rolipram, a specific and well-known inhibitor of PDE4.
The effects of Rolipram in the experimental systems (in vitro
and in vivo) used by Park et al.—for example, cAMP increase,
AMPK activation, mitogenesis—overlap nicely with those produced by resveratrol, and this is a strong piece of evidence
supporting the mechanistic outline. Rolipram is being considered for intervention in a range of pathophysiological situations, including inflammation, mood disorders, neurodegeneration, memory enhancement, immunomodulation, metabolic
syndrome. Importantly, in vivo Rolipram is used at doses 200
times lower than is resveratrol: its metabolism differs radically
from that of resveratrol, with a much greater persistence of
the unaltered drug in the circulatory stream.[23]
So, do we have a synthetic “improved resveratrol” that
might eliminate the need to drink wine? Perhaps not quite
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yet. The effects of Rolipram in vivo have been extensively investigated, and their desirability seems to depend on circumstances. It has side effects (vomiting), and a clinical trial studying its potential against multiple sclerosis was interrupted,
owing to safety concerns, because the drug was poorly tolerated.[24] We might be better off with natural compounds. Interestingly, genistein (a soy isoflavone) and other polyphenols are
PDE inhibitors with affinities comparable to that of resveratrol.[25]
Park and co-workers focus on the mechanisms of resveratrol
action channelled through AMPK and Sirt1 activation and impinging on cellular metabolism, bioenergetics, autophagy and
on conditions such as obesity or diabetes. As usual, the advances made open up a host of other perspectives. How do the
activation of Rap small GTPases, modulation of the Ca2 + signal
and activation of Sirt1 relate to the other major (putative)
activities of resveratrol, in particular protection of the cardiovascular system and cancer prevention/therapy? For example
Raps (a family within the Ras superfamily), are regulators of cytoskeletal organisation and integrin activation, with important
roles in chemokine-induced lymphocyte migration and integrin-mediated adhesion[26] and in the regulation of various parameters in the cardiovascular system.[27] In the heart, they
participate in Ca2 + signalling and excitation–contraction. In the
microvasculature, they can act to maintain vessel integrity in
the face of cellular stress. Could Rap activation contribute to
the much talked about effects of resveratrol on the circulatory
system?
While aspects such as these remain to be investigated, the
topic of extending life by using this phytochemical continues
to be at the centre of public attention. Does the work by Park
and colleagues offer new hope by pointing at previously unknown players of the game (such as PDE4) that might become
targets of anti-aging intervention? Alas, the proposal that Sirt1
activation results in an extension of lifespan downstream of CR
is not holding up well to scrutiny. (e.g., ref. [28]). Still, evidence
from transgenic mice indicates it might have a protective
effect against several age-associated conditions and pathologies[28a] (by the way: how can a protein have an impact on
age-related disorders but not on lifespan?). Perhaps other sirtuins are involved. Sirt3, one of the mitochondrial sirtuins, has
been reported to be of importance in the context of CR and
aging.[5b, 29] It is proposed to act mainly by reducing ROS generation by mitochondria.[29b] The effects of phytochemicals on
this enzyme have not been extensively investigated yet, but
a recent report suggests that resveratrol might actually decrease its expression.[30] The life-lengthening effect of resveratrol in model organisms has also come to be doubted.[8, 28c, 31] It
has never been claimed for healthy mammals kept on a regular
diet. The evidence endures, however, that resveratrol has positive effects, including life extension, for obese mice.[31]
So in the end, if not the fountain of youth, perhaps a “slimness pill”. At least, and at last, we now know how.
2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 2012, 13, 1256 – 1259
Resveratrol and Health
Acknowledgements
The authors’ work is supported by the National Research Council
of Italy and by the Fondazione Cassa di Risparmio di Padova
e Rovigo (CARIPARO) (Excellence grant “Developing a pharmacology of polyphenols”).
Keywords: aging · AMPK · phosphodiesterases · resveratrol ·
rolipram
[1] a) J. M. Smoliga, J. A. Baur, H. A. Hausenblas, Mol. Nutr. Food Res. 2011,
55, 1129 – 1141; b) K. R. Patel, E. Scott, V. A. Brown, A. J. Gescher, W. P.
Steward, K. Brown, Ann. N. Y. Acad. Sci. 2011, 1215, 161 – 169.
[2] S. J. Park, F. Ahmad, A. Philp, K. Baar, T. Williams, H. Luo, H. Ke, H. Rehmann, R. Taussig, A. L. Brown, M. K. Kim, M. A. Beaven, M. B. Burgin, V.
Manganiello, J. J. Chung, Cell 2012, 148, 421 – 433.
[3] Y. Hu, J. Liu, J. Wang, Q. Liu, Free Radical Biol. Med. 2011, 51, 250 – 256.
[4] C. J. Kenyon, Nature 2010, 464, 504 – 512.
[5] a) G. Donmez, L. Guarente, Aging Cell 2010, 9, 285 – 290; b) A. Chalkiadaki, L. Guarente, Nat. Rev. Endocrinol. 2012, 8, 287 – 296.
[6] J. A. Palacios, D. Herranz, M. L. De Bonis, S. Velasco, M. Serrano, M. A.
Blasco, J. Cell Biol. 2010, 191, 1299 – 1313.
[7] K. T. Howitz, K. J. Bitterman, H. Y. Cohen, D. W. Lamming, S. Lavu, J. G.
Wood, R. E. Zipkin, P. Chung, A. Kisielewski, L. L. Zhang, B. Scherer, D. A.
Sinclair, Nature 2003, 425, 191 – 196.
[8] M. Kaeberlein, T. McDonagh, B. Heltweg, J. Hixon, E. A. Westman, S. D.
Caldwell, A. Napper, R. Curtis, P. S. DiStefano, S. Fields, A. Bedalov, B. K.
Kennedy, J. Biol. Chem. 2005, 280, 17038 – 17045.
[9] a) C. Cant, J. Auwerx, Cell. Mol. Life Sci. 2010, 67, 3407 – 3423; b) C.
Cant, J. Auwerx, Pharmacol. Rev. 2012, 64, 166 – 187.
[10] C. Cant, J. Auwerx, Curr. Opin. Lipidol. 2009, 20, 98 – 105.
[11] a) B. M. Emerling, F. Weinberg, C. Snyder, Z. Burgess, G. M. Mutlu, B. Viollet, G. R. Budinger, N. S. Chandel, Free Radical Biol. Med. 2009, 46, 1386 –
1391; b) L. Chen, B. Xu, L. Liu, J. Yin, H. Zhou, W. Chen, T. Shen, X. Han,
S. Huang, Lab. Invest. 2010, 90, 762 – 773.
[12] C. Cant, Z. Gerhart-Hines, J. N. Feige, M. Lagouge, L. Noriega, J. C.
Milne, P. J. Elliott, P. Puigserver, J. Auwerx, Nature 2009, 458, 1056 –
1060.
[13] a) B. Dasgupta, J. Milbrandt, Proc. Natl. Acad. Sci. USA 2007, 104, 7217 –
7222; b) V. Vingtdeux, L. Giliberto, H. Zhao, P. Chandakkar, Q. Wu, J. E.
Simon, E. M. Janle, J. Lobo, M. G. Ferruzzi, P. Davies, P. Marambaud, J.
Biol. Chem. 2010, 285, 9100 – 9113; c) J. H. Um, S. J. Park, H. Kang, S.
Yang, M. Foretz, M. W. McBurney, M. K. Kim, B. Viollet, J. H. Chung, Diabetes 2010, 59, 554 – 563.
ChemBioChem 2012, 13, 1256 – 1259
[14] C. Cant, L. Q. Jiang, A. S. Deshmukh, C. Mataki, A. Coste, M. Lagouge,
J. R. Zierath, J. Auwerx, Cell Metab. 2010, 11, 213 – 219.
[15] S. Chung, H. Yao, S. Caito, J. W. Hwang, G. Arunachalam, I. Rahman,
Arch. Biochem. Biophys. 2010, 501, 79 – 90.
[16] T. Szkudelski, K. Szkudelska, Ann. N. Y. Acad. Sci. 2011, 1215, 34 – 39.
[17] S. M. Shin, I. J. Cho, S. G. Kim, Mol. Pharmacol. 2009, 76, 884 – 895.
[18] a) J. Zheng, V. D. Ramirez, Br. J. Pharmacol. 2000, 130, 1115 – 1123; b) R.
Zini, C. Morin, A. Bertelli, A. A. Bertelli, J. P. Tillement, Drugs Exp. Clin.
Res. 1999, 25, 87 – 97.
[19] S. P. Collins, J. L. Reoma, D. M. Gamm, M. D. Uhler, Biochem. J. 2000, 345,
673 – 689.
[20] a) E. Wenzel, V. Somoza, Mol. Nutr. Food Res. 2005, 49, 472 – 481; b) T.
Walle, Ann. N. Y. Acad. Sci. 2011, 1215, 9 – 15.
[21] a) X. Vitrac, A. Desmoulire, B. Brouillaud, S. Krisa, G. Deffieux, N. Barthe,
J. Rosenbaum, J. M. Mrillon, Life Sci. 2003, 72, 2219 – 2233; b) M.
Abd El-Mohsen, H. Bayele, G. Kuhnle, G. Gibson, E. Debnam, S. Kaila Srai,
C. Rice-Evans, J. P. Spencer, Br. J. Nutr. 2006, 96, 62 – 70; c) M. E. Juan, M.
Maij, J. M. Planas, J. Pharm. Biomed. Anal. 2010, 51, 391 – 398.
[22] F. Visioli, C. A. De La Lastra, C. Andres-Lacueva, M. Aviram, C. Calhau, A.
Cassano, M. D’Archivio, A. Faria, G. Fav, V. Fogliano, R. Llorach, P. Vitaglione, M. Zoratti, M. Edeas, Crit. Rev. Food Sci. Nutr. 2011, 51, 524 – 546.
[23] W. Krause, G. Khne, N. Sauerbrey, Eur. J. Clin. Pharmacol. 1990, 38, 71 –
75.
[24] B. Bielekova, N. Richert, T. Howard, A. N. Packer, G. Blevins, J. Ohayon,
H. F. McFarland, C. S. Strzebecher, R. Martin, Mult. Scler. 2009, 15,
1206 – 1214.
[25] a) W. C. Ko, C. M. Shih, Y. H. Lai, J. H. Chen, H. L. Huang, Biochem. Pharmacol. 2004, 68, 2087 – 2094; b) C. H. Shih, L. H. Lin, Y. H. Lai, C. Y. Lai,
C. Y. Han, C. M. Chen, W. C. Ko, Eur. J. Pharmacol. 2010, 643, 113 – 120.
[26] D. S. Johnson, Y. H. Chen, Curr. Opin. Pharmacol. 2012, 12, 1 – 6.
[27] S. C. Jeyaraj, N. T. Unger, M. A. Chotani, Life Sci. 2011, 88, 645 – 652.
[28] a) D. Herranz, M. Serrano, Nat. Rev. Cancer 2010, 10, 819 – 823; b) C. Burnett, S. Valentini, F. Cabreiro, M. Goss, M. Somogyvri, M. D. Piper, M.
Hoddinott, G. L. Sutphin, V. Leko, J. J. McElwee, R. P. Vazquez-Marinque,
A. M. Orfila, D. Ackerman, C. Au, G. Vinti, M. Riesen, K. Howard, C. Neri,
A. Bedalov, M. Kaeberlein, C. Soti, L. Partridge, D. Gems, Nature 2011,
477, 482 – 485; c) J. Couzin-Frankel, Science 2011, 334, 1194 – 1198.
[29] a) O. M. Palacioos, J. J. Carmona, S. Michan, K. Y. Chen, Y. Manabe, J. L.
Ward, 3rd, L. J. Goodyear, Q. Tong, Aging 2009, 1, 771 – 783; b) E. L. Bell,
L. Guarente, Mol. Cell 2011, 42, 561 – 568.
[30] H. Schirmer, T. C. Pereira, E. P. Rico, D. B. Rosemberg, C. D. Bonan, M. R.
Bogo, A. A. Souto, Mol. Biol. Rep. 2012, 39, 3281 – 3289.
[31] B. Agarwal, J. A. Baur, Ann. N. Y. Acad. Sci. 2011, 1215, 138 – 143.
Received: March 20, 2012
Published online on May 13, 2012
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