The expression dynamics of transforming growth factor-β/Smad signaling in the liver fibrosis experimentally caused by Clonorchis sinensis
- Chao Yan†1,
- Lin Wang†1,
- Bo Li1,
- Bei-Bei Zhang1,
- Bo Zhang1,
- Yan-Hong Wang1,
- Xiang-Yang Li1,
- Jia-Xu Chen2,
- Ren-Xian Tang1Email author and
- Kui-Yang Zheng1Email author
© Yan et al,; licensee BioMed Central. 2015
Received: 16 December 2014
Accepted: 20 January 2015
Published: 4 February 2015
Liver fibrosis is a hallmark of clonorchiasis suffered by millions people in Eastern Asian countries. Recent studies showed that the activation of TGF-β/Smad signaling pathway can potently regulate the hepatic fibrogenesis including Schistosoma spp. and Echinococcus multilocularis-caused liver fibrosis. However, little is known to date about the expression of transforming growth factor-β (TGF-β) and other molecules in TGF-β/Smad signaling pathway which may play an important role in hepatic fibrosis caused by C. sinensis.
A total of 24 mice were individually infected orally with 45 metacercariae, both experimental mice and mocked-infected control mice were anesthetized at 4 week post-infection (wk p.i.), 8 wk p.i. and 16 wk p.i., respectively. For each time-point, the liver and serum from each animal were collected to analyze histological findings and various fibrotic parameters including TGF-β1, TGF-β receptors and down-stream Smads activation, as well as fibrosis markers expression.
The results showed that collagen deposition indicated by hydroxyproline content and Masson’s trichrome staining was increased gradually with the development of infection. The expression of collagen type α1 (Col1a) mRNA transcripts was steadily increased during the whole infection. The mRNA levels of Smad2, Smad3 as well as the protein of Smad3 in the liver of C. sinensis-infected mice were increased after 4 wk p.i. (P < 0.05, compared with normal control) whereas the TGF-β1, TGF-β type I receptor (TGFβRI) and TGF-β type II receptor (TGFβRII) mRNA expression in C. sinensis-infected mice were higher than those of normal control mice after 8 wk p.i. (P < 0.05). However, the gene expression of Smad4 and Smad7 were peaked at 4 wk p.i. (P < 0.05), and thereafter dropped to the basal level at 8 wk p.i., and 16 wk p.i., respectively. The concentrations of TGF-β1 in serum in the C. sinensis-infected mice at 8 wk p.i. and 16 wk p.i (P < 0.05) were significantly higher than those in the control mice.
The results of the present study indicated for the first time that the activation of TGF-β/Smad signaling pathway might contribute to the synthesis of collagen type I which leads to liver fibrosis caused by C. sinensis.
Clonorchis sinensis is a food-borne zoonotic parasite, which is epidemic in some Eastern Asian countries including China, Korea, Japan, and Vietnam. In humans, it is assumed that approximately 15–20 million people were suffering from clonorchiasis whereas the number of infected people was 12.5 million in China according to a report based on a nationwide survey [1,2]. Human become infected by ingestion of freshwater fish containing C. sinensis metacercariae. The metacercariae develop into C. sinensis juveniles in the duodenum by the stimulation of trypsin, and then rapidly move to the intrahepatic bile duct where the juvenile worms become mature and survive for decades [3,4]. C. sinensis infection can induce significant cholangitis, adenomatous hyperplasia mechanical obstruction of the hepatobiliary duct and cholelithiasis . Furthermore, C. sinensis is considered as a group I carcinogen-metazoan parasite to potentially induce cholangiocarcinoma in humans . Chronic infection with C. sinensis can also potently lead to liver fibrosis which is marked with excessive accumulation of extracellular matrix components (ECM) due to an imbalance between its synthesis and degradation [2,7,8]. Moreover, some components of worms and its excretory/secretory products (ESP) which can probably participate in the development of hepatic fibrosis have been widely investigated in the lab of Professor Yu [9-13]. However, molecular mechanism underlying fibrotic responses of hosts to these virulence factors is not fully elucidated.
Transforming growth factor-β (TGF-β) as one of major pro-fibrotic cytokines plays a crucial role in orchestrating fibrogenesis and it is demonstrated that active TGF-β1 motivates its downstream signaling pathway, leading to phosphorylation of Smad2 and Smad3 (also called R-Smad) which is meditated by TGF-β type I (TGFβRI) and type II receptors (TGFβRII), phosphorylated Smad2 and Smad3 rapidly combine with a common mediator called Smad4 and subsequently migrates to the nucleus, resulting in massive fibrotic genes expression (such as collagen type I) [14-17]. TGF-β/Smad signaling pathway has been proved as a canonical pathway that can potently regulate the hepatic fibrogenesis [18,19], and a few studies have addressed about the activation of TGF-β/Smad signaling in fibrogenesis caused by parasitic infection, such as Schistosoma spp. and Echinococcus multilocularis, which suggested that TGF-β/Smad signaling play important roles in the development of liver fibrosis [20-22]. However, to our best knowledge, little is known of the expression and potential roles of TGF-β/Smad signaling pathway which may be involved in process of hepatic fibrosis caused by C. sinensis. In the light of this background, the objectives of the present study were to investigate the expression dynamics of TGF-β/Smad pathway and analyze their possible roles in the development of hepatic fibrosis in BALB/c mice infected by C. sinensis.
Pseudorasbora parva, the second intermediate hosts which were naturally infected with C. sinensis, were collected in Guangxi Autonomous Region, People’s Republic of China. And the fish were transported to our laboratory by air. Metacercariae of C. sinensis were collected by digesting fish with a pepsin-HCl (0.6%) artificial gastricjuice. The collected metacercariae were preserved in cold Alsever’s solution with antibiotics until use.
Female BABL/c mice (6 ~ 8 weeks old, 22 ± 2 g) were purchased from Shanghai Laboratory Animal Co., Ltd (SLAC, Shanghai, China). The mice were housed in an air-conditioned room at 24°C with a 12 h dark/light cycle and permitted free access to standard laboratory food and water. All animal experiments were approved by the Animal Care and Use Committee of Xuzhou Medical Collage. The mice were individually infected orally with 45 metacercariae. Mock-infected control mice were similarly administered with 50 μl of sterile normal solution. Both experimental mice (n = 24) and control mice (n = 15) were divided into 3 groups and anesthetized at 4 week post-infection (wk p.i.), 8 wk p.i. and 16 wk p.i., respectively. For each time-point, the liver and serum from each animal were harvested to analyze histological findings and various fibrotic parameters.
Histological examination and evaluation of hepatic fibrosis in mice caused by C. sinensis
For histological evaluation, all liver samples were fixed in formalin, embedded in paraffin. 4 μm thick sections were prepared and then stained with hematoxylin and eosin (H&E) and Masson’s trichrome (MT). These specimens were observed and photographed under an inverted microscope. Collagen depositions from 5–8 images of each specimen were quantified using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).
Determination of hydroxyproline content
Collagen was also determined by evaluating the concentration of the hydroxyproline (Hyp), an amino acid characteristic of collagen. The lysates were used to measure hydroxyproline contents using commercially available kits according to the manufacturer’s instructions (Jiancheng Institute of Biotechnology, Nanjing, China). In this kit, hydroxyproline concentration was determined by the reaction of oxidized hydroxyproline with 4-(Dimethylamino) benzaldehyde (DMAB), which was measured spectrophotometrically at 560 nm.
Quantitative real-time PCR analysis
Primers used in the present study
Expected size (bp)
Total protein was extracted from liver tissues and analyzed with bicinchoninic acid protein concentration assay kit (Beyotime Biotech, Beijing, China). Sample protein was separated by electrophoresis in 12% SDS-PAGE with a Bio-Rad electrophoresis system (Hercules, CA, USA). The primary antibodies (rabbit anti-Smad3 antibody, UCallM biotech Co., Ltd, Wuxi, China, 1:1000 dilutions) were incubated at 4°Covernight. The corresponding horseradish-peroxidase-conjugated secondary antibodies (anti-rabbit IgG, 1:5000 dilutions) were incubated for 1 h at room temperature. The membrane containing antibody-protein complexes were visualized with an enhanced chemiluminescence detection system on radiograph film. The brands were scanned and analyzed by the software Quantity ONE (Bio-rad, Hercules, CA, USA). The expression of protein in each sample was normalized by α-Tublin(Santa Cruz Biotechnology, CA, USA).
Enzyme linked immunosorbent assay (ELISA)
Serum from each mouse was immediately used to evaluate the concentration of TGF-β1 by a specific ELISA kit (eBiosciences, CA, USA). In brief, samples were firstly activated by 1 mol/L HCl, and then samples as well as serial dilutions of standards were added to 96-well plates pre-coated with anti-TGF-β1 and pre-blocked with PBS containing 10% fetal bovine serum (FBS), after samples were washed, horseradish peroxidase (HRP)-conjugated streptavidin A in PBS containing 10% FBS was added for 30 min at room temperature. After final washes, the HRP substrate TMB (3,3,5,5-tetramethylbenzidine) was added, and the optical density of the color reaction was measured at 450 nm. Concentrations of cytokine in the sera were calculated using standard curves as references.
All values were expressed as mean ± SEM. Comparisons between control and each experimental group were made by one-way analysis of variance (ANOVA) and Student’s unpaired t-test using the SPSS 13.0 statistical package. Differences were considered statistically significant at P < 0.05.
Histological findings and evaluation of hepatic fibrosis in mice caused by C. sinensis
The hydroxyproline which is a major component of collagen was also evaluated as an indicator of collagen content. Compared with normal control mice, the levels of hydroxyproline were significantly augmented at 4 wk p.i. (P < 0.001), and thereafter dramatically increased at 8 wk p.i. (P < 0.001) and 16 wk p.i. (P < 0.001, Figure 1C).
The expression of the pro-fibrotic molecular markers in livers of mice during C. sinensis infection
The mRNA expression of TGF-β/Smad in the liver of BALB/c during C. sinensis-infection
The protein expression of Smad3 in livers of C. sinensis-infected mice and dynamic changes of serous TGF-β1 in mice infected by C. sinensis
As shown in Figure 4C, during the whole experimental infection, the concentration of TGF-β1 in the serum at 8 wk p.i. and 16 wk p.i. was significantly higher than that of in the control mice (P < 0.05), and the level of TGF-β1 were increased from 4 wk p.i. to 16 wk p.i during the development of infection.
Previous studies showed that liver fibrosis was orchestrated by a complex network of signaling pathways involved in regulation the deposition of extracellular matrix, and of these signaling pathways, TGF-β/Smad signaling pathway is considered as the most prominent mediator in accelerating liver fibrosis [18,23]. Beside TGF-β, IL-13 and Il-17 were recently demonstrated as another critical pro-fibrotic cytokines in liver fibrosis, for example, IL-13 can potently induce the synthesis of collagen I and other fibrotic markers directly in Schistosoma spp. caused liver fibrosis whereas IL-17A can promote the activation of HSC and drive the mRNA expression of the IL-6, α-SMA, collagen, as well as TGF-β1 in carbon tetrachloride–induced liver fibrosis [24-27]. However, little is known of the molecular mechanism underlying C. sinensis caused liver fibrosis. In the present study, we used BALB/c mice to explore the possible mechanism underlying the liver fibrosis caused by C. sinensis. Similar with other study, we showed that BALB/c mice can develop a moderate periductal fibrosis at 4 wk p.i. and massive deposition of extracellular matrix after 8 wk p.i. demonstrated by HE and MT staining, suggesting that the mouse model for C. sinensis induced-liver fibrosis in the present study was established successfully [7,8].
Biological functions of TGF-β and its roles in regulating ECM deposition have been intensively reviewed, and proteins of the Smads family members are important mediators that transduce signals induced by TGF-β to specific target genes in the nucleus, leading to the expression of pro-fibrotic genes [28,29]. There were also some studies suggesting that TGF-β1 and its downstream Smads played a central role in parasite-induced liver fibrosis [20,30,31], for example, in Schistosoma mansoni-infected mouse, the increased expression of TGF-β1 and its receptors led to extensive accumulation of extracellular matrix proteins and treatment with anti-fibrotic drugs like praziquantel can reduce the concentration of TGF-β signicantly and led to an reversible liver fibrosis in S. mansoni-infected mice [32,33].
As expected, hepatic mRNA transcriptions of fibrotic markers such as collagen type I (Col1a), TGF-β1, α-SMA and hydroxyproline contents were increased with the degree of C. sinensis-caused hepatic fibrosis, which was indicated by MT staining. To further investigate whether TGF-β/Smad signaling was activated during C. sinensis-caused liver fibrosis or not, the expression of genes and proteins within TGF-β/Smad signaling pathway was examined. In the present study, the mRNA expression of TGF-β1, Smad2/3, TGFβRI and TGFβRII was significantly stronger in the livers of C. sinensis infected-mice than that in control ones, and these changes were positively correlated with the degrees of hepatic fibrosis (data is not shown), suggesting that TGFβ/Smad signaling pathway may be involved in the development of liver fibrosis due to C. sinensis infection. However, our study showed Smad4 was found increased at 4 wk p.i., and its expression decreased with the development of hepatic fibrosis, which suggested that the effects of Smad4 might occur at early and middle stage of hepatic fibrosis . Our results also showed that Smad7 expression heightened only at the 4 wk p.i. and the expression of Smad2 was significantly higher in the livers of C. sinensis-infection mice, indicating that Smad7 played a negative role in fine-tuning of TGF-β signals since the increased Smad2 may suppress the expression of Smad7 conversely [35,36].
In conclusion, our results of the present study suggested, for the first time, that expression dynamics of TGF-β/Smad signaling pathway may be involved in the development of hepatic fibrosis caused by C. sinensis. The further studies should be warranted to elucidate detailed roles of TGF-β/Smad signaling pathway in C. sinensis caused liver fibrosis, which may provide basic information for control clonorchiasis.
Project support was provided, in part, the National Natural Science Foundation of China (Grant Nos. 81171590 and 31302077), the Open Funds of the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Grant No. SKLVEB2013KFKT005), the Graduate Innovation Program in Science and Technology of Jiangsu Province, China (Grant No. KYLX14-1448). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
- Fang YY, Chen YD, Li XM, Wu J, Zhang QM, Ruan CW. Current prevalence of Clonorchis sinensis infection in endemic areas of China. ZhongguoJi Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 2008, 26:99–103, 109.Google Scholar
- Hong ST, Fang Y. Clonorchis sinensis and clonorchiasis, an update. Parasitol Int. 2012;61:17–24.View ArticlePubMedGoogle Scholar
- Li S, Chung YB, Chung BS, Choi MH, Yu JR, Hong ST. The involvement of the cysteine proteases of Clonorchis sinensis metacercariae in excystment. Parasitol Res. 2004;93:36–40.View ArticlePubMedGoogle Scholar
- Kim TI, Yoo WG, Kwak BK, Seok JW, Hong SJ. Tracing of the Bile-chemotactic migration of juvenile Clonorchis sinensis in rabbits by PET-CT. PLoS Negl Trop Dis. 2011;5:e1414.View ArticlePubMed CentralPubMedGoogle Scholar
- Chen J, Xu MJ, Zhou DH, Song HQ, Wang CR, Zhu XQ. Canine and feline parasitic zoonoses in China. Parasit Vectors. 2012;5:152.View ArticlePubMed CentralPubMedGoogle Scholar
- Bouvard V, Baan R, Straif K, Grosse Y, Secretan B, El Ghissassi F, et al. A review of human carcinogens–Part B: biological agents. Lancet Oncol. 2009;10:321–2.View ArticlePubMedGoogle Scholar
- Yoon BI, Choi YK, Kim DY, Hyun BH, Joo KH, Rim HJ, et al. Infectivity and pathological changes in murine clonorchiasis: comparison in immunocompetent and immunodeficient mice. J Vet Med Sci. 2001;63:421–5.View ArticlePubMedGoogle Scholar
- Choi YK, Yoon BI, Won YS, Lee CH, Hyun BH, Kim HC, et al. Cytokine responses in mice infected with Clonorchis sinensis. Parasitol Res. 2003;91:87–93.View ArticlePubMedGoogle Scholar
- Wang X, Hu F, Hu X, Chen W, Huang Y, Yu X. Proteomic identification of potential Clonorchis sinensis excretory/secretory products capable of binding and activating human hepatic stellate cells. Parasitol Res. 2014;113:3063–71.View ArticlePubMedGoogle Scholar
- Liang P, Sun J, Huang Y, Zhang F, Zhou J, Hu Y, et al. Biochemical characterization and functional analysis of fructose-1,6-bisphosphatase from Clonorchis sinensis. Mol Biol Rep. 2013;40:4371–82.View ArticlePubMedGoogle Scholar
- Zhang F, Liang P, Chen W, Wang X, Hu Y, Liang C, et al. Stage-specific expression, immunolocalization of Clonorchissinensis lysophospholipase and its potential role in hepatic fibrosis. Parasitol Res. 2013;112:737–49.View ArticlePubMedGoogle Scholar
- Zheng M, Hu K, Liu W, Hu X, Hu F, Huang L, et al. Proteomic analysis of excretory secretory products from Clonorchis sinensis adult worms: molecular characterization and serological reactivity of a excretory-secretory antigen-fructose-1,6-bisphosphatase. Parasitol Res. 2011;109:737–44.View ArticlePubMedGoogle Scholar
- Hu F, Hu X, Ma C, Zhao J, Xu J, Yu X. Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A(2) and investigation of its potential contribution to hepatic fibrosis. Mol Biochem Parasitol. 2009;167:127–34.View ArticlePubMedGoogle Scholar
- Kanzler S, Lohse AW, Keil A, Henninger J, Dienes HP, Schirmacher P, et al. TGF-beta1 in liver fibrosis: an inducible transgenic mouse model to study liver fibrogenesis. Am J Physiol. 1999;276:1059–68.Google Scholar
- Dooley S, Delvoux B, Streckert M, Bonzel L, Stopa M, ten Dijke P, et al. Transforming growth factor beta signal transduction in hepatic stellate cells via Smad2/3 phosphorylation, a pathway that is abrogated during in vitro progression to myofibroblasts. TGFbeta signal transduction during transdifferentiation of hepatic stellate cells. FEBS Lett. 2001;502:4–10.View ArticlePubMedGoogle Scholar
- Schiller M, Javelaud D, Mauviel A. TGF-beta-induced SMAD signaling and gene regulation: consequences for extracellular matrix remodeling and wound healing. J Dermatol Sci. 2004;35:83–92.View ArticlePubMedGoogle Scholar
- Yoshida K, Murata M, Yamaguchi T, Matsuzaki K. TGF-β/Smad signaling during hepatic fibro-carcinogenesis (review). Int J Oncol. 2014;45:1363–71.PubMed CentralPubMedGoogle Scholar
- Date M, Matsuzaki K, Matsushita M, Sakitani K, Shibano K, Okajima A, et al. Differential expression of transforming growth factor-beta and its receptors in hepatocytes and nonparenchymal cells of rat liver after CCl4 administration. J Hepatol. 1998;28:572–81.View ArticlePubMedGoogle Scholar
- Yoshida K, Matsuzaki K. Differential regulation of TGF-β/Smad signaling in hepatic stellate cells between acute and chronic liver injuries. Front Physiol. 2012;3:53.View ArticlePubMed CentralPubMedGoogle Scholar
- Wang J, Zhang C, Wei X, Blagosklonov O, Lv G, Lu X, et al. TGF-β and TGF-β/Smad signaling in the interactions between Echinococcus multilocularis and its hosts. PLoS One. 2013;8:e55379.View ArticlePubMed CentralPubMedGoogle Scholar
- Zhang BB, Cai WM, Tao J, Zheng M, Liu RH. Expression of Smad proteins in the process of liver fibrosis in mice infected with Schistosoma japonicum. Zhong guo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2013;31:89–94.Google Scholar
- Chen BL, Peng J, Li QF, Yang M, Wang Y, Chen W. Exogenous bone morphogenetic protein-7 reduces hepatic fibrosis in Schistosomajaponicum-infected mice via transforming growth factor-β/Smad signaling. World J Gastroenterol. 2013;19:1405–15.View ArticlePubMed CentralPubMedGoogle Scholar
- Bataller R, Brenner DA. Liver fibrosis. J Clin Invest. 2005;115:209–18.View ArticlePubMed CentralPubMedGoogle Scholar
- Liu Y, Munker S, Müllenbach R, Weng HL. IL-13 signaling in liver fibrogenesis. Front Immunol. 2012;3:116.View ArticlePubMed CentralPubMedGoogle Scholar
- Chuah C, Jones MK, Burke ML, McManus DP, Gobert GN. Cellular and chemokine-mediated regulation in schistosome-induced hepatic pathology. Trends Parasitol. 2014;30:141–50.View ArticlePubMedGoogle Scholar
- Tang J, Huang H, Ji X, Zhu X, Li Y, She M, et al. Involvement of IL-13 and tissue transglutaminase in liver granuloma and fibrosis after schistosoma japonicum infection. Mediators Inflamm. 2014;2014:753483.PubMed CentralPubMedGoogle Scholar
- Tan Z, Qian X, Jiang R, Liu Q, Wang Y, Chen C, et al. IL-17A plays a critical role in the pathogenesis of liver fibrosis through hepatic stellate cell activation. J Immunol. 2013;191:1835–44.View ArticlePubMedGoogle Scholar
- Chen N, Geng Q, Zheng J, He S, Huo X, Sun X. Suppression of the TGF-β/Smad signaling pathway and inhibition of hepatic stellate cell proliferation play a role in the hepatoprotective effects of curcumin against alcohol-induced hepatic fibrosis. Int J Mol Med. 2014;34:1110–6.PubMedGoogle Scholar
- Dooley S, ten Dijke P. TGF-β in progression of liver disease. Cell Tissue Res. 2012;347:245–56.View ArticlePubMed CentralPubMedGoogle Scholar
- Zhu D, He X, Duan Y, Chen J, Wang J, Sun X, et al. Expression of microRNA-454 in TGF-β1-stimulated hepatic stellate cells and in mouse livers infected with Schistosoma japonicum. Parasit Vectors. 2014;7:148.View ArticlePubMed CentralPubMedGoogle Scholar
- El-Lakkany NM, Hammam OA, El-Maadawy WH, Badawy AA, Ain-Shoka AA, Ebeid FA. Anti-inflammatory/anti-fibrotic effects of the hepatoprotectivesilymarin and the schistosomicide praziquantel against Schistosoma mansoni-induced liver fibrosis. Parasit Vectors. 2012;5:9.View ArticlePubMed CentralPubMedGoogle Scholar
- Barros AF, Oliveira SA, Carvalho CL, Silva FL, de Souza VC, da Silva AL, et al. Low transformation growth factor-β1 production and collagen synthesis correlate with the lack of hepatic periportal fibrosis development in undernourished mice infected with Schistosoma mansoni. Mem Inst Oswaldo Cruz. 2014;109:210–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Attia YM, Elalkamy EF, Hammam OA, Mahmoud SS, El-Khatib AS. Telmisartan, an AT1 receptor blocker and a PPAR gamma activator, alleviates liver fibrosis induced experimentally by Schistosoma mansoni infection. Parasit Vectors. 2013;6:199.View ArticlePubMed CentralPubMedGoogle Scholar
- Wang HQ, Ding XD, Wu Q, Zhang Q, Huang Y, Yang F. Expression of transforming growth factor–beta and Smads in hepatic fibrosis induced by Schistosoma Japonica in mice. World J Gastroenterol. 2008;16:929–34.Google Scholar
- Sobral LM, Montan PF, Zecchin KG, Martelli-Junior H, Vargas PA, Graner E, et al. Smad7 blocks transforming growth factor-β1-induced gingival fibroblast-myofibroblast transition via inhibitory regulation of Smad2 and connective tissue growth factor. J Periodontol. 2011;82:642–51.View ArticlePubMedGoogle Scholar
- Kamiya Y, Miyazono K, Miyazawa K. Smad7 inhibits transforming growth factor-beta family type i receptors through two distinct modes of interaction. J Biol Chem. 2010;285:30804–13.View ArticlePubMed CentralPubMedGoogle Scholar
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.