- Open Access
Vaticanol C, a resveratrol tetramer, activates PPARα and PPARβ/δ in vitro and in vivo
- Tomoko Tsukamoto†1,
- Rieko Nakata†1,
- Emi Tamura1,
- Yukiko Kosuge1,
- Aya Kariya1,
- Michiko Katsukawa1,
- Satoshi Mishima2,
- Tetsuro Ito3,
- Munekazu Iinuma3,
- Yukihiro Akao4,
- Yoshinori Nozawa4,
- Yuji Arai5,
- Shobu Namura6 and
- Hiroyasu Inoue1Email author
© Tsukamoto et al; licensee BioMed Central Ltd. 2010
Received: 16 March 2010
Accepted: 27 May 2010
Published: 27 May 2010
Appropriate long-term drinking of red wine is associated with a reduced risk of cardiovascular disease. Resveratrol, a well-known SIRT1 activator is considered to be one of the beneficial components contained in red wine, and also developed as a drug candidate. We previously demonstrated that resveratrol protects brain against ischemic stroke in mice through a PPARα-dependent mechanism. Here we report the different effects of the oligomers of resveratrol.
We evaluated the activation of PPARs by ε-viniferin, a resveratrol dimer, and vaticanol C, a resveratrol tetramer, in cell-based reporter assays using bovine arterial endothelial cells, as well as the activation of SIRT1. Moreover, we tested the metabolic action by administering vaticanol C with the high fat diet to wild-type and PPARα-knockout male mice for eight weeks.
We show that vaticanol C activates PPARα and PPARβ/δ in cell-based reporter assays, but does not activate SIRT1. ε-Viniferin shows a similar radical scavenging activity as resveratrol, but neither effects on PPARs and SIRT-1. Eight-week intake of vaticanol C with a high fat diet upregulates hepatic expression of PPARα-responsive genes such as cyp4a10, cyp4a14 and FABP1, and skeletal muscle expression of PPARβ/δ-responsive genes, such as UCP3 and PDK4 (pyruvate dehydrogenase kinase, isoform 4), in wild-type, but not PPARα-knockout mice.
Vaticanol C, a resveratrol tetramer, activated PPARα and PPARβ/δ in vitro and in vivo. These findings indicate that activation of PPARα and PPARβ/δ by vaticanol C may be a novel mechanism, affording beneficial effects against lifestyle-related diseases.
Prevention of lifestyle-related diseases, such as cardiovascular disease, diabetes, and stroke, has become of worldwide interest. In such lifestyle-related matters, people pay attention not only to drugs, but also to the functionality of natural chemicals contained in food and drink, such as polyphenols and their polymers. In this context, resveratrol, a phytoalexin and antioxidant polyphenol included in red wine and various plants, is one of the most attractive compounds, and experimental data on resveratrol has been accumulating [1, 2]. The polymers of resveratrol are also known phytoalexins and antioxidants. The resveratrol dimer, ε-viniferin, is a major phytoalexin in response to UV-C irradiation  and has been reported to be an antioxidant . The resveratrol tetramer, vaticanol C, exhibited a more potent cytotoxic effect against human cultured cancer cells than resveratrol [5–7]. By screening 20 derivatives of resveratrol, vaticanol C was found to have the strongest suppressive activity of cell growth in colon cancer cell lines . However, the molecular mechanisms for these actions remain to be determined.
We demonstrated that resveratrol activates peroxisome proliferator-activated receptor (PPAR) α and γ in cell-based reporter assays and protects the brain against ischemic stroke in mice through a PPARα-dependent mechanism . PPARs are members of nuclear receptor family of ligand-dependent transcription factors . The PPAR subfamily consists of three isotypes, PPARα, β/δ, and γ, which play various roles in lipid and carbohydrate metabolism, cell proliferation and differentiation, and inflammation and are considered to be molecular targets against lifestyle-related diseases [10, 11]. For example, PPARα and γ agonists, such as fibrates and thiazolidine derivatives, are used to treat dyslipidemia and diabetes, respectively. Moreover, eicasapentaenoic acid, a natural ligand for PPARα, has been used as a hypolipidemic drug and has been reported to lower plasma and liver cholesterol levels in a PPARα-dependent manner . Thus, we have focused on PPARs as possible molecular targets of resveratrol in preventing lifestyle-related diseases, while others have studied SIRT1, a NAD+-dependent protein deacetylase, and PGC-1α [13–17].
In the present study, we evaluated the activation of PPARs, antioxidant, and SIRT1-activator properties of resveratrol, ε-viniferin, and vaticanol C and found that vaticanol C activates PPARα and PPARβ/δ in cell-based reporter assays, but does not have antioxidant or SIRT1-activator properties. Moreover, 8-week intake of vaticanol C with a high fat (HF) diet upregulates hepatic expression of PPARα-responsive genes and skeletal muscle expression of PPARβ/δ-responsive genes in wild-type, but not PPARα-knockout mice. These findings indicate that activation of PPARα and PPARβ/δ by vaticanol C may provide beneficial effects against lifestyle-related diseases.
ε-Viniferin, a resveratrol dimmer, and vaticanol C, a resveratrol tetramer, were isolated from the genus belonging to Vatica, Shorea, and Vateria (Dipterocarpaceae) as described previously [5, 6]. A stock solution of these compounds was prepared in 10 mM dimethyl sulfoxide (DMSO), and was further diluted to the working concentration before use.
RNA Extraction and Analysis
Total RNA was isolated using the acid guanidinium thiocyanate procedure, and was analyzed for gene expression via real-time quantitative RT-PCR (Mx3005, Stratagene) as described previously . The primer pairs for genes in this study and their cycling conditions are shown in Additional file 1. Expression levels of each mRNA were normalized to those of 36B4 mRNA.
In the case of activation assay for PPARα, BAEC was transfected with 0.15 μg of tk-PPREx3-Luc reporter plasmid, 0.15 μg of human PPARα expression vector pGS-hPPARα (Invitrogen, GeneStorm™ clone L02932) and 0.02 μg of pSV-βgal as described previously using Trans IT-LT-1 (Mirus) . In the activation assay for PPARβ/δ and γ, their expression vectors pCMX-hNUCI (PPAR β/δ) and pCMX-hPPARγ1 were used for the transfection, instead of the human PPARα expression vector . The transfected BAEC were harvested, lysed, and assayed for both luciferase and β-galactosidase activities. The results are represented as relative luciferase activities, which were normalized against the β-galactosidase standard. Transfection efficiency of BAEC is over 20% estimated by cotransfection of pEGFP-N1, an expression vector for Green Fluorescent Protein. pCMX-hNUCI and pCMX-hPPARγ1 were human PPARγ1 and PPARβ/δ expression vectors under control of a cytomegalovirus promoter . In the positive control experiments, selective activators of PPARs, such as Wy-14643 (α), GW501516 (β/δ), and pioglitazone (γ), had PPARα, β/δ, and γ agonistic activities, respectively .
DPPH Radical Scavenging and SIRT1 Activation Assays
Resveratrol and its derivatives were dissolved in DMSO to prepare samples at 500 μM. Twenty microliters of each sample, 80 μL of 0.1 M Tris HCl buffer (pH 7.4), and 100 μL of 500 μM 1,1-diphenyl-2-picryl-hydrazyl (DPPH) solution were mixed well and incubated at room temperature for 20 min, then the absorbance was recorded at 540 nm with a plate reader (Immuno mini NJ2300, Nalgene) as described . Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) was the reference antioxidant, used as a positive control, and each result is expressed as the equivalent per mole of trolox from the percentage decrease with respect to the negative control value. Data are presented as mean ± SD (n = 3).
The Fluor de Lys fluorescence assay for in vitro SIRT1 activity was performed by the SIRT1 activity assay kit (BIOMOL, catalog number AK-555), according to the manufacturer's instructions. Fluorescent intensity was measured using an Infinite F200 microplate fluorometer (TECAN). DMSO was used as a negative control and also as a solvent for resveratrol and its derivatives to a final concentration of 100 μM in the assay buffer.
Male 12-week-old 129SV-strain (wild-type) and PPARα-knockout (129SV background) mice were obtained by Jackson laboratory (USA). These mice were housed in a room at 24 ± 2°C with a 12/12 h light-dark cycle, and fed a HF diet containing 60% energy as fat, supplemented with vaticanol C (0 and 0.04%) for 8 weeks. Diet and water were available ad libitum. After 8 weeks of feeding, the mice were sacrificed under diethyl ether anesthesia to obtain tissues. Liver and skeletal muscle were stored in RNAlater™ solution (Ambion, USA) at -30°C until gene expression analysis. These experimental procedures were approved by the Animal Care Committee of Nara Women's University.
All results are expressed as means ± SD. Comparisons between groups were performed with the unpaired t test. Values were deemed to be statistically significantly different at p < 0.05.
Activation of PPARs In Vitro by Resveratrol and Vaticanol C but not by ε-Viniferin
No Correlation with Resveratrol or its Derivatives between Activation of PPARs and Radical-Scavenging Activities
Next, we evaluated resveratrol and its derivatives for their radical-scavenging activity using DPPH, a stable free radical . As shown in Figure 2B, resveratrol and ε-viniferin showed similar radical-scavenging activities, comparable to the positive control radical scavenger, trolox, a water-soluble derivative of vitamin E. On the other hand, vaticanol C showed no radical scavenging activity (Figure 2B), indicating that there was no correlation between agonistic activities for PPARs and radical-scavenging activity.
No Activation of SIRT1 by Vaticanol C or ε-Viniferin
Because resveratrol has been reported to be an activator for SIRT1 , we evaluated the activation of SIRT1 by ε-viniferin and vaticanol C. As a positive control, resveratrol activated SIRT1; however, ε-viniferin and vaticanol C did not (Figure 2C). Rather, vaticanol C seemed to inhibit SIRT1 activity in this assay. Taken together, vaticanol C, a resveratrol tetramer, is a dual agonist for PPARα and β/δ, but not a radical scavenger and not a SIRT1 activator.
Vaticanol C Administration Induces Expression of PPAR-dependent Genes
The metabolic actions of vaticanol C were then evaluated by administering the compound by food admixture (0 and 0.04%) to wild-type and PPARα-knockout male mice challenged with the HF diet for 8 weeks. During this experiment, there was no statistically significant difference in body weight between the eight groups.
Resveratrol is known to be a SIRT1-activator. However, various effects of resveratrol are not explained by activation of SIRT1 alone. We found that resveratrol is an activator not only for PPARα and γ , but also for PPARβ/δ (Figure 1B) in this report. Moreover, vaticanol C, a resveratrol tetramer, is an activator for PPARα and β/δ in cell-based reporter assays (Figure 1B), which is confirmed by induced expression of PPAR-responsive genes in wild-type, but not PPARα-knockout, mice (Figure 3A). At lower concentrations of 1.25-5 μM, vaticanol C showed higher PPARα-agonistic activity than resveratrol. However, at 10 μM, resveratrol showed higher activity than vaticanol C (Figure 1A), and this activity of resveratrol was dose-dependent, at less than 40 μM (data not shown). Previously, we found that expression of COX-2, an inducible key enzyme for prostaglandin synthesis, was regulated by 15d-PGJ2, a natural ligand for PPARγ . Interestingly, vaticanol C as well as resveratrol  suppressed the expression of COX-2 in several kinds of tumor cell lines (data not shown) whereas vaticanol C is not an activator for PPARγ.
Vaticanol C did not activate SIRT1 whereas resveratrol activated SIRT1 by the assay utilizing a fluorophore-containing peptide (Figure 2C). Recent report showed that this assay was not suitable to direct measurement of the SIRT1 activity and that resveratrol did not activate SIRT1 in the assay without the fluorophore-containing peptide . Therefore, by the different assay system, we may confirm that vaticanol C is not activator for SIRT1. Taken together, vaticanol C is a dual activator for PPARα and β/δ, whereas resveratrol is a triple activator for PPARα, β/δ and γ, indicating that vaticanol C is thought to be differently effective on lifestyle-related diseases, compared with resveratrol.
A resveratrol dimer, ε-viniferin has radical-scavenging activity, but does not have agonistic activity for PPARs or SIRT1, which differs from resveratrol and vaticanol C (Figure 2). These differences indicate the importance of the structure of these chemicals. Concerning the binding pocket of PPARα, the molecular sizes of resveratrol and vaticanol C are smaller and larger than those of synthetic PPAR agonists such as fenofibrate, respectively. Resveratrol but not vaticanol C activates PPAR γ, indicating the importance of the structure of these molecules in the binding pocket of PPARs. It was reported that a single amino acid, which was Tyr in PPARα and His in PPARγ, imparted subtype selectivity for both thiazolidinedione and nonthiazolidinedione ligands . We need more experimental data to discuss about these points including the possibility of indirect activation of PPARs by resveratrol and vaticanol C.
There are distinct expression patterns of PPAR-responsive genes in the liver and skeletal muscle, indicating that PPAR-responsive genes are controlled in a tissue-specific manner (Figure 3A). In this context, especially in skeletal muscles, vaticanol C upregulates expression of PPARβ/δ-responsive genes, such as UCP3 and PDK4, in the wild-type, but not PPARα-knockout mice, indicating that PPARα is also involved in the expression of PPARβ/δ-responsive genes.
Resveratrol has been reported to prevent body weight gain with a HF diet . No such effect on body weight was observed after vaticanol C treatment. We found that 129SV mice used in our study showed milder body weight gain with a HF diet, versus the C57/black strain mice used in several reports. This difference on body weight gain will be due to the different strains between 129SV and C57/black. These genetic differences are also usual common in human genomes. To address these questions, we are studying on the molecular mechanism involved in the physiological differences between 129SV and C57/black mice.
Vaticanol C, a resveratrol tetramer, activates PPARα and PPARβ/δ in cell-based reporter assays, but does not activates SIRT1. Eight-week intake of vaticanol C with a HF diet upregulates hepatic expression of PPARα-responsive genes and skeletal muscle expression of PPARβ/δ-responsive genes in wild-type but not PPARα-knockout mice. These findings indicate that activation of PPARα and PPARβ/δ by vaticanol C is a novel mechanism that may afford beneficial effects against cardiovascular disease.
We thank Dr. Takanobu Matsubara, Haruka Takeuchi, Mariko Hotta, and Kanako Kojima for technical assistance. This work was supported by Grants-in-Aid for Scientific Research (Nos. 15310158 to HI and 19300250 to HI and RN) from the Ministry of Education, Culture, Sports, Science and Technology, Japan. SN was supported by NS048532 from NIH/NINDS, USA.
- Lastra CA, Villegas I: Resveratrol as an anti-inflammatory and anti-aging agent: mechanisms and clinical implications. Mol Nutr Food Res. 2005, 49: 405-430. 10.1002/mnfr.200500022.View ArticleGoogle Scholar
- Baur JA, Sinclair DA: Therapeutic potential of resveratrol: the in vivo evidence. Nat Rev Drug Discov. 2005, 5: 493-506. 10.1038/nrd2060.View ArticleGoogle Scholar
- Douillet-Breuil AC, Jeandet P, Adrian M, Bessis R: Change in the phytoalexin contert of various vitis spp. in response to ultraviolet C elicitation. J Agric Food Chem. 1999, 47: 4456-4461. 10.1021/jf9900478.View ArticleGoogle Scholar
- Privat C, Telo JP, Bernardes-Genisson V, Vieira A, Souchard JP, Nepveu F: Antioxidant properties of trans-epsilon-viniferin as component to stilibane derivatives in aqueous and nonaqueous media. J Agric Food Chem. 2002, 50: 1213-1217. 10.1021/jf010676t.View ArticleGoogle Scholar
- Ito T, Akao Y, Tanaka T, Iinuma M, Nozawa Y: Vaticanol C, a novel resveratrol tetramer, inhibits cell growth through induction of apoptosis in cancer cell lines. Biol Pharm Bull. 2002, 25: 147-148. 10.1248/bpb.25.147.View ArticleGoogle Scholar
- Ito T, Akao Y, Yi H, Ohguchi K, Matsumoto K, Tanaka T, Iinuma M, Nozawa Y: Antitumor effect of resveratrol oligomers against human cancer cell lines and the molecular mechanism of apoptosis induced by vaticanol C. Carcinogenesis. 2003, 24: 1489-1497. 10.1093/carcin/bgg105.View ArticleGoogle Scholar
- Shibata MA, Akao Y, Shibata E, Nozawa Y, Ito T, Mishima S, Morimoto J, Otsuki Y: Vaticanol C, a novel resveratrol tetramer, reduces lymph node and lung metastates of mouse mammary carcinoma carrying p53 mutation. Cancer Chemother Pharmacol. 2007, 60: 681-691. 10.1007/s00280-007-0414-y.View ArticleGoogle Scholar
- Inoue H, Jiang X, Katayama T, Osada S, Umesono K, Namura S: Brain protection by resveratrol and fenofibrate against stroke requires peroxisome proliferator-activated receptor alpha in mice. Neurosci Lett. 2003, 352: 203-206. 10.1016/j.neulet.2003.09.001.View ArticleGoogle Scholar
- Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M, Chambon P: The nuclear receptor superfamily: the second decade. Cell. 1995, 83: 835-839. 10.1016/0092-8674(95)90199-X.View ArticleGoogle Scholar
- Michalik L, Auwerx J, Berger JP, Chatterjee VK, Glass GK, Gonzalez FJ, Grimaldi PA, Kadowaki T, Lazar MA, O'Rahilly S, Palmer CN, Plutzky J, Reddy JK, Spiegelman BM, Staels B, Wahli W: International Union of Pharmacology. LX1. Peroxisome proliferators- acitivated receptors. Pharmacol Rev. 2006, 58: 726-741. 10.1124/pr.58.4.5.View ArticleGoogle Scholar
- Sonoda J, Pei L, Evans RM: Nuclear receptors: decoding metabolic disease. FEBS Lett. 2008, 9: 2-9. 10.1016/j.febslet.2007.11.016.View ArticleGoogle Scholar
- Sugiyama E, Ishikawa Y, Li Y, Kagai T, Nobayashi M, Tanaka N, Kamijo Y, Yokoyama S, Hara A, Aoyama T: Eicosapentaenoic acid lowers plasma and liver cholesterol levels in the presence of peroxisome proliferators-activated receptor alpha. Life Sci. 2008, 83: 19-28. 10.1016/j.lfs.2008.04.011.View ArticleGoogle Scholar
- Howitz KT, Bitterman KJ, Cohen HY, Lamming DW, Lavu S, Wood JG, Zipkin RE, Chung P, Kisielewski A, Zhang LL, Scherer B, Sinclair DA: Small molecule activators of sirtuins extend Saccharomyces cerevisiae. Nature. 2003, 425: 191-196. 10.1038/nature01960.View ArticleGoogle Scholar
- Baur JA, Pearson KJ, Price NL, Jamieson HA, Lerin C, Kalra A, Prabhu VV, Allard JS, Lopez-Lluch G, Lewis K, Pistell PJ, Poosala S, Becker KG, Boss O, Gwinn D, Wang M, Ramaswamy S, Fishbein KW, Spencer RG, Lakatta EG, Le Couteur D, Shaw RJ, Navas P, Puigserver P, Ingram DK, de Cabo R, Sinclair DA: Resveratrol improves health and survival of mice on a high-caloric diet. Nature. 2006, 444: 337-342. 10.1038/nature05354.View ArticleGoogle Scholar
- Lagouge M, Argmann C, Gerhart-Hines Z, Meziane H, Lerin C, Daussin F, Messadeq N, Milne J, Lambert P, Elliott P, Geny B, Laakso M, Puigserver P, Auwerx J: Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1alpha. Cell. 2006, 127: 1109-1122. 10.1016/j.cell.2006.11.013.View ArticleGoogle Scholar
- Feige JN, Lagouge M, Canto C, Strehle A, Houten SM, Milne JC, Lambert PD, Mataki C, Elliott PJ, Auwerx J: Specific SIRT1 activation mimics lower energy levels and protects against diet-induced metabolic disorders by enhancing fat oxidation. Cell Metab. 2008, 8: 347-358. 10.1016/j.cmet.2008.08.017.View ArticleGoogle Scholar
- Pfluger PT, Herranz D, Velasco-Miguel S, Serrano M, Tschöp MH: Sirt1 protects against high-fat diet-induced metabolic damage. Proc Natl Acad Sci. 2008, 105: 9793-9798. 10.1073/pnas.0802917105.View ArticleGoogle Scholar
- Hotta M, Nakata R, Katsukawa M, Hori K, Takahashi S, Inoue H: Carvacrol, a component of thyme oil, activates PPARα and γ, and suppresseses COX-2 expression. J Lipid Res. 2010, 51: 132-139. 10.1194/jlr.M900255-JLR200.View ArticleGoogle Scholar
- Inoue H, Tanabe T, Umesono K: Feedback control of cyclooxygenase-2 expression through PPAR gamma. J Biol Chem. 2000, 275: 28028-28032.Google Scholar
- Fauconneau B, Waffo-Teguo P, Huguet F, Barrier L, Decendit A, Merillon JM: Comparative study of radical scavenger and antioxidant properties of phenolic components from Vitis vinifera cell cultures using in vitro tests. Life Sci. 1997, 61: 2103-2110. 10.1016/S0024-3205(97)00883-7.View ArticleGoogle Scholar
- Inagaki T, Dutchak P, Zhao G, Ding X, Gautron L, Parameswara V, Li Y, Goetz R, Mohammadi M, Esser V, Elmquist JK, Gerard RD, Burgess SC, Hammer RE, Mangelsdorf DJ, Kliewer SA: Endocrine regulation of the fasting response by PPARα-mediated induction of fibroblast growth factor 21. Cell Metab. 2007, 5: 415-425. 10.1016/j.cmet.2007.05.003.View ArticleGoogle Scholar
- Badman MK, Pissios P, Kennedy AR, Koukos G, Flier JS, Maratos-Flier E: Hepatic fibroblast growth factor 21 is regulated by PPARα and is a key mediator of hepatic metabolism in ketotic states. Cell Metab. 2007, 5: 426-437. 10.1016/j.cmet.2007.05.002.View ArticleGoogle Scholar
- Son C, Hosoda K, Matsuda J, Fujikura J, Yonemitsu S, Iwakura H, Masuzaki H, Ogawa Y, Hayashi T, Itoh H, Nishimura H, Inoue G, Yoshimasa Y, Yamori Y, Nakao K: Up-regulation of uncoupling protein 3 gene expression by fatty acid and agonists for PPARs in L6 myotubes. Endocrinology. 2001, 142: 4189-4194. 10.1210/en.142.10.4189.View ArticleGoogle Scholar
- Tanaka T, Yamamoto J, Iwasaki S, Asaba H, Hamura H, Ikeda Y, Watanabe M, Magoori K, Ioka RX, Tachibana K, Watanabe Y, Uchiyama Y, Sumi K, Iguchi H, Ito S, Doi T, Hamakubo T, Naito M, Auwerx J, Yanagisawa M, Kodama T, Sakai J: Activations of peroxisome proliferators- activated receptor delta induces fatty acid beta-oxidation in skeletal muscle and attenuates metabolic syndrome. Proc Natl Acad Sci. 2003, 100: 15924-15929. 10.1073/pnas.0306981100.View ArticleGoogle Scholar
- Subbaramaiah WJ, Chung K, Michaluart P, Telang N, Tanabe T, Inoue H, Jang M, Pezzuto JM, Dannenberg AJ: Resveratrol inhibits cyclooxygenase-2 transcription and activity in phorbol ester-treated human mammary epithelial cells. J Biol Chem. 1998, 273: 21875-2188227. 10.1074/jbc.273.34.21875.View ArticleGoogle Scholar
- Pacholec M, Bleasdale JM, CHrunyk B, Chunningham D, Flynn D, Garofalo RS, Griffith D, Griffor M, Loulakis P, Pabst B, Qui X, Stockman B, Thanabal V, Varghese A, Ward J, Withka J, Ahn K: SRT1720, SRT2183, SRT1460, and resveratrol are not direct activators of SIRT1. J Biol Chem. 2010, 285: 8340-8351. 10.1074/jbc.M109.088682.View ArticleGoogle Scholar
- Xu HE, Lambert MH, Montana VG, Plunket KD, Moore LB, Collins JL, Oplinger JA, Kliewer SA, Gample RT, Mckee DD, Moore JT, Willson TM: Structural determinants of ligand binding selectivity between the peroxisome proliferator-activated receptors. Proc Natl Acad Sci. 2001, 98: 13919-13924. 10.1073/pnas.241410198.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.