ORIGINAL RESEARCH

BASIC SCIENCE

Taurine Supplementation Improves Erectile Function in Rats withStreptozotocin-induced Type 1 Diabetes via Amelioration of PenileFibrosis and Endothelial Dysfunction

Yajun Ruan, MD,1,2 Mingchao Li, MD, PhD,1,2 Tao Wang, MD, PhD,1,2 Jun Yang, MD, PhD,1,2 Ke Rao, MD, PhD,1,2

Shaogang Wang, MD, PhD,1,2 Weiming Yang, MD, PhD,1,2 Jihong Liu, MD, PhD,1,2 and Zhangqun Ye, MD, PhD1,2

ABSTRACT

Received Se1DepartmenUniversity o2Institute oUniversity

Copyright ªElsevier Inc.http://dx.doi

778

Introduction: For patients with diabetes, erectile dysfunction (ED) is common and greatly affects quality of life.However, these patients often exhibit a poor response to first-line oral phosphodiesterase type 5 inhibitors.

Aim: To investigate whether taurine, a sulfur-containing amino acid, affects diabetic ED (DED).

Methods: Type 1 diabetes mellitus was induced in male rats by using streptozotocin. After 12 weeks, anapomorphine test was conducted to confirm DED. Only rats with DED were administered taurine or vehicle for4 weeks. Age-matched nondiabetic rats were administered saline intraperitoneally for 4 weeks.

Main Outcome Measures: Erectile function was evaluated by electrical stimulation of the cavernous nerve.Histologic and molecular alterations of the corpus cavernosum also were analyzed.

Results: Erectile function was significantly reduced in the diabetic rats compared with in the nondiabetic rats,and was improved in the diabetic rats treated with taurine. The corpus cavernosum of the rats with DEDexhibited severe fibrosis and decreased smooth muscle content. Deposition of extracellular matrix proteins wasincreased in the diabetic rats, while expression of endothelial nitric oxide synthase/cyclic guanosine mono-phosphate/nitric oxide pathwayerelated proteins was reduced. Taurine supplementation ameliorated erectileresponse as well as histologic and molecular alterations.

Conclusion: Taurine supplementation improves erectile function in rats with DED probably by potentialantifibrotic activity. This finding provides evidence for a potential new therapy for DED.

J Sex Med 2016;13:778e785. Copyright � 2016, International Society for Sexual Medicine. Published by ElsevierInc. All rights reserved.

Key Words: Taurine; Diabetes; Erectile Dysfunction; Fibrosis; Extracellular Matrix

INTRODUCTION

Erectile dysfunction (ED), defined as the inability to attain ormaintain a penile erection sufficient for successful vaginalintercourse, is becoming more common worldwide.1 Approxi-mately 15% to 20% of the general male population experiencesED.2,3 Patients with diabetes are nearly 3 times more likely tosuffer from ED compared to patients without diabetes. Inaddition, ED appears to arise 10 years earlier in patients withdiabetes and tends to be more severe, significantly decreasing

ptember 25, 2015. Accepted February 17, 2016.

t of Urology, Tongji Hospital, Tongji Medical College, Huazhongf Science and Technology, Hubei, China;

f Urology, Tongji Hospital, Tongji Medical College, Huazhongof Science and Technology, Hubei, China

2016, International Society for Sexual Medicine. Published byAll rights reserved..org/10.1016/j.jsxm.2016.02.164

health-related quality of life.4 Furthermore, the prevalence of EDis 95% in patients with diabetes above the age of 60.3

Introduction of phosphodiesterase type 5 (PDE5) inhibitorsrevolutionized the treatment of ED. PDE5 inhibitors enhancenitric oxide (NO)-mediated relaxation of the corpus cavernosumby preventing cyclic guanosine monophosphate (cGMP)degradation. Currently, PDE5 inhibitors are the first-line therapyfor ED.5e6 However, because ED is caused by multiple factors, acertain proportion of patients, especially those with diabetes,exhibit a poor response to PDE5 inhibitors.7 Moreover, the failurerate for all PDE5 inhibitors is higher in men with diabetes than inmen without.8 In addition, diabetes could induce a series ofpathophysiologic changes that could contribute to the decreasedresponse to PDE5 inhibitors. Therefore, it is urgent to developnovel treatment modalities for men with diabetic ED (DED).

Taurine (2-aminoethanesulfonic acid), a sulfur-containingamino acid, is one of the most abundant free amino acids in the

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Taurine Improves Diabetic Erectile Dysfunction via Anti-fibrosis 779

human body. It plays a role in several physiologic functions,including neuromodulation or neurotransmission, modulation ofcalcium flow, osmoregulation, stabilization of membranes, andbile formation in the liver.9 Data from existing studies have indi-cated that taurine might decrease cholesterol level, control highblood pressure, and have protective potential in the cardiovascularsystem.10 Previous studies also have shown a beneficial effect oftaurine on diabetes-associated complications. Importantly, it hasbeen demonstrated that taurine supplementation can enhancesexual response and mating ability in aged rats.11 However,whether taurine could mitigate DED has not been investigated.

In the present study, we evaluated the effect of taurine in ratswith DED in vivo. Our results show that taurine improveserectile function in diabetic rats and could serve as an alternativetherapy for ED.

METHODS

Treatment of AnimalsThis study was approved by the Animal Care and Use Com-

mittee of Tongji Hospital, Tongji Medical College, HuazhongUniversity of Science and Technology, Hubei, China. Forty-five 8-week-old male Sprague-Dawley rats were used in the present study.After overnight fasting, the rats were injected with freshly preparedstreptozotocin (60 mg/kg; Sigma-Aldrich, St Louis, MO, USA;n ¼ 37) or vehicle (0.1 mol/L citrate-phosphate buffer; pH, 4.2;n¼ 8; nondiabetic control group) intraperitoneally. Blood glucoselevels weremeasured 72 hours after streptozotocin injection using ablood glucosemeter (ACCU-CHEKPerforma;RocheDiagnostics,Shanghai, China). Only rats with a fasting glucose concentration�16.7 mmol/L were considered as diabetic rats (n ¼ 35).

At 12 weeks, 30 diabetic rats survived. An apomorphine(APO) test was conducted to confirm DED. Eighteen APO-negative rats were used for further experiments.12,13 Rats withDED were administered taurine (400 mg/kg; Sigma-Aldrich;DED þ taurine group) or vehicle (saline, DED group) intra-peritoneally daily for another 4 weeks (n ¼ 9 per group). Thisdose of taurine is within the range used in previous studies.14e16

In Vivo Erection StudiesAt 12 weeks after diabetes was induced, erectile function was

evaluated by observing APO-induced erection in all survivingrats. APO was injected into the loose skin of the cervical vertebrasubcutaneously (80 mg/kg). Then a camera was set in the bottomof the cage for 30 minutes, and the number of erections wasrecorded. The criteria for penile erection were based on previousstudies as follows: stretching of the penis with a congested glans,repeated pelvic thrusts immediately followed by an uprightposition, and complete emergence of an engorged glans penisand distal penile shaft; otherwise, the result was negative.13,17

At week 16, following a 2-day washout of taurine, intra-cavernosal pressure (ICP) was measured in all groups as describedpreviously.18 Under proper anesthesia (pentobarbital sodium, 40

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mg/kg, i.p.), the rats were fixed on the table, and the left carotidartery was exposed. A PE-50 tube filled with heparinized saline(100 IU/mL) was cannulated into the artery and connected to apressure transducer to monitor mean arterial pressure (MAP)continuously. A 25-G needle was inserted into the left penile crurato monitor ICP. Pressure data were recorded by a data acquisitionsystem (PowerLab/4SP; AD Instruments, Bella Vista, Australia).Erectile response was elicited by electrical stimulation using abipolar electrode hooked to the cavernous nerve. Stimulation pa-rameters were as follows: 7.5 v, 12 Hz, and 1.2 ms; this was judgedto cause a maximal reaction.18e20 The ratio between maximumICP and MAP (ICP max/MAP) was calculated to normalize forvariations in systemic blood pressure. Total ICPwas determined bythe area under the curve (AUC, mmHg $ s) during stimulation.

After finishing these in vivo studies, the rats were sacrificed byintraperitoneal overdose of pentobarbital. Penile midshafts weremaintained in 4% paraformaldehyde overnight and embedded inparaffin for subsequent histologic studies. Remaining penile tis-sues were snap frozen in liquid nitrogen and stored at �80�C forsubsequent molecular detection. Blood samples were obtainedfrom rats via the vena cava. The total testosterone levels weremeasured in the central laboratory of our institution.

Western Blot AnalysisEqual amounts (40 mg/lane) of protein extracts were electro-

phoresed on 10% sodium dodecylsulfateepolyacrylamide gels,transferred to polyvinylidene fluoride membranes (Immobilon-PTransferMembrane; Millipore Corporation, Billerica, MA, USA),and probed with antibodies against endothelial nitric oxide syn-thase (eNOS; 1:500; BD Biosciences, San Jose, CA, USA), plas-minogen activator inhibitor type 1 (PAI-1; 1:2500; BDBiosciences), collagen I (1:500; Boster, Wuhan, China), collagenIV (1:500; Proteintech, Wuhan, China), transforming growthfactor b1 (TGF-b1; 1:500; Abcam, Cambridge, UK), aesmoothmuscle actin (a-SMA; 1:1000; Abcam), or b-actin (1:500; Boster).Primary antibodies were incubated overnight at 4�C followed byincubation with horseradish peroxidase-labeled secondary anti-body (1:10,000; Jackson ImmunoResearch, West Grove, PA,USA), then detected with Bio-Rad ClarityWestern ECL Substrate(Bio-Rad Laboratories, CA, USA). The resulting image wasanalyzed using ImageJ software (http://rsb.info.nih.gov/ij) todetermine the integrated density value of each protein band.

cGMP MeasurementcGMP was measured in extracts from penile samples using a

commercial cGMP enzyme immunoassay kit (Cayman Chem-icals Inc, Ann Arbor, MI, USA) following the manufacturer’sinstructions. Protein content in each sample was measured usingthe BCA assay and expressed as range of pmol cGMP/mgprotein. All samples were analyzed in triplicate.20

Histologic AssessmentPenile tissue sections (5 mm thickness) were processed for

immunohistochemistry as described previously.21 The primary

Table 1. Metabolic and physiological variables

Variable Controla (n ¼ 8) DEDa (n ¼ 9) DED þ taurinea (n ¼ 9)

Initial weight (g) 240.90 ± 11.47 233.10 ± 11.09 233.00 ± 12.06Final weight (g) 522.90 ± 60.31 243.00 ± 42.66* 257.50 ± 33.35*Initial fasting glucose (mmol/L) 6.25 ± 0.41 6.12 ± 0.44 6.11 ± 0.48Final fasting glucose (mmol/L) 6.23 ± 0.47 30.54 ± 2.48* 30.86 ± 2.11*Testosterone (ng/mL) 2.55 ± 0.61 0.73 ± 0.05* 1.60 ± 0.25*,†

MAP (mm Hg) 112.50 ± 9.73 114.10 ± 10.30 115.80 ± 10.56ICP (mm Hg) 97.58 ± 9.04 43.28 ± 12.10*,† 71.99 ± 16.08*AUC (mm Hg $ s) 5125 ± 723.6 1504 ± 505.2*,† 3687 ± 1308*

MAP ¼ mean arterial pressure; ICP ¼ intracavernous pressure; AUC ¼ area under the curve.a Data are presented as mean value ± standard deviation.* P< .05 compared with the control group.† P< .05 compared with the DED þ taurine group.

780 Ruan et al

antibody was mouse antiea-SMA (1:150; Boster). Masson’strichrome staining was performed according to the standardprotocol. Semiquantitative analysis was performed to evaluateintensity using Image-Pro plus software (Media Cybernetics,Silver Spring, MD, USA).

Statistical AnalysisResults were analyzed using GraphPad Prism version 5.0

(GraphPad Software, San Diego, CA, USA) and expressed asmean ± standard error of the mean. Statistical analyses wereperformed using 1-way analysis of variance followed by theTukey-Kramer test for post hoc comparisons. Differences amonggroups were considered significant at P < .05.

RESULTS

Metabolic Variables and ICP/MAP AssessmentThere were no significant differences in initial body weight or

serum glucose concentration among the 3 groups. At 16 weeksafter diabetes was induced, fasting glucose concentration wassignificantly higher, and body weight along with total testos-terone levels were significantly lower in the diabetic rats than inthe age-matched normal controls. No significant differences inbody weight or glucose concentration were found between theDED and DED þ taurine groups (Table 1).

Erectile function was evaluated by measuring maximum ICP,ICP max/MAP, and total ICP. All erectile function variableswere significantly lower in the diabetic rats than in the controlrats (Figure 1). Partial but significant recovery of erectile functionwas seen in the DED þ taurine group. MAP did not differsignificantly among the groups (Table 1, Figure 1).

Extracellular Matrix Protein ProductionWe performed Western blot analysis to evaluate production of

extracellular matrix (ECM) proteins, including PAI-1, collagen I,and collagen IV. Cavernous ECM protein production wassignificantly higher in the DED group than in the normal con-trols (P < .05); however, taurine reversed this change (Figure 2).

We also performed Western blot analysis to evaluate expres-sion of a-SMA and TGF-b1 in each group. The diabetic ratsshowed lower a-SMA expression than the normal controls.Expression of a-SMA was higher in the DED þ taurine groupthan in the DED group (all P < .05; Figures 4B and 4E).Meanwhile, expression of TGF-b1 was higher in the DED groupthan in the normal controls; however, taurine partially reversedits upregulation.

eNOS ExpressionWe performed eNOS Western blot analysis to quantify

endothelial function in all groups. Penile tissue in the rats withDED showed significantly lower eNOS immunoexpressioncompared with the normal controls (P < .05). This decline wasattenuated in part by taurine (Figures 3A and 3B).

cGMP ConcentrationCavernous tissue cGMP concentration in the DED group was

significantly lower than that in the normal controls (P < .05).The DED þ taurine group had a higher concentration of cGMPthan the DED group (P < .05; Figure 3C).

Histologic ChangesAs displayed in Figure 4, corporal tissue in the diabetic rats

showed a significantly lower smooth muscle/collagen ratiocompared with tissue in the normal controls (0.13 and 0.30,respectively; P < .05). Taurine increased the ratio in theDED þ taurine group compared with that in the DED group.Immunohistochemical staining showed that taurine-treatedDED group had a significant increase of a-SMA in corpuscavernosum compared with DED group (13.53% and 7.73%;P < .05).

DISCUSSION

In this study, we demonstrated that administration of taurine,not as a food supplement, in rats with streptozotocin-induceddiabetes significantly decreased penile fibrosis; significantly

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Figure 1. Increase of erectile function in taurine-treated diabetic rats. (A) Representative tracing of intracavernosal pressure (ICP) andmean arterial pressure (MAP) for age-matched normal control, diabetic erectile dysfunction (DED) and taurine-treated DED groups. (B, C)Erectile function presented as maximum ICP/MAP and total ICP (AUC) in each group. *P < .05 compared with the control group. †P < .05compared with the DED þ Taurine group.

Taurine Improves Diabetic Erectile Dysfunction via Anti-fibrosis 781

decreased production of ECM proteins, including PAI-1,collagen I, and collagen IV; and upregulated eNOS expression;and that these changes were associated with physiologicallyrelevant changes in erectile function.Taurine is known to play a role in a variety of cellular processes,

including calcium regulation, inflammation, osmoregulation,oxidative stress, and cysteine detoxification, all of which may

Figure 2. Taurine reduces cavernous extracellular matrix protein depinhibitor type1 (PAI-1, 47 kDa), transforming growth factor beta 1 (TGFcontrol group, DED group and DED rats treated with taurine group. (B)with that of b-actin. *P < .05 compared with the control group. †P <

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contribute to cardiovascular homeostasis. Taurine has become apotential therapeutic target for cardiovascular disease, preventingstroke, atherosclerosis, and hypertension.22Maia et al reported thattaurine supplementation was beneficial to control high bloodpressure and vascular remodeling induced via postweaning proteinrestriction.16 Besides, the protective effect of taurine on endothelialfunction in diabetic rats already has been proved.14 As a benchmark

osition. (A) Representative Western blot for plasminogen activator-b1, 37 kDa), collagen I (139 kDa), and collagen IV (161 kDa) in theData are presented as the relative density of each protein compared.05 compared with the DED þ Taurine group.

Figure 3. Taurine treatment restores cavernous endothelial nitric oxide synthase (eNOS) expression. (A) Representative Western blot foreNOS (140 kDa) in each group. (B) Data are presented as the relative density values of eNOS to the b-actin loading control. The relativeratio measures in the control group is arbitrarily presented as 1. (C) ELISA demonstrating quantity of cGMP. *P < .05 compared with thecontrol group. †P < .05 compared with the DED þ Taurine group.

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for cardiovascular disease,23 erectile function also has been inves-tigated. A previous ex vivo study indicated that chronic treatmentwith taurine could prevent development of corpus cavernosumdysfunction after induction of diabetes.15 Dalaklioglu et al foundthat the protective effect of taurine might be associated with sup-pression of nicotinamide adenine dinucleotide phosphate oxidaseand Rho-kinase activity. However, the NO/cGMP pathway,which is the most classic pathway in the erectile process, has notbeen discussed. Our results suggest that taurine may partiallyrestore endothelial function via upregulation of the eNOS/cGMPpathway. Our present work reproduced previous findingsdemonstrating improved NO production and/or bioavailability,and extended that knowledge by suggesting an antifibrosis effect ofthis amino acid in the penis.

Fibrosis is involved in most diabetic complications, such ascardiomyopathy and nephropathy. Corporal fibrosis also hasemerged as the predominant underlying cause of DED, especiallyin poor responders to PDE5 inhibitors. Although it differs in or-gans and cells, fibrosis shares some common characteristics. Inpatients with diabetes, hyperglycemic conditions may serve as theinitial insult throughout the corpora and penile arteries, which istypically followed by an inflammatory response.24 Profibroticfactors are subsequently released. It has been demonstrated inanimal models that diabetes leads to increased expression of TGF-b1 and penile fibrosis.25e26 These studies suggest that upregula-tion of TGF-b1 and its downstream effectors might play a pivotalrole in diabetes-induced structural changes.25e27 In line with these

previous studies, our work suggests that in diabetic rats, TGF-b1and PAI-1 are both upregulated. It is well documented that TGF-b1 has multiple biologic functions, including cell proliferation,differentiation, apoptosis, autophagy, and ECM protein produc-tion.28 The role of TGF-b1 in penile fibrosis has been confirmedby measuring mRNA expression in penile tissues of young andaging rats.29 TGF-b1 expression was higher in older rats than inyoung rats. In diabetes models, accumulation of myofibroblastsand the switch to a synthetic phenotype producing ECM proteinsdue to TGF-b1 activation have been shown to exacerbate penilefibrosis. As such, collagen and ECM proteins accumulate in thepenis, accompanied by loss of functional cells. PAI-1, the mostpotent cellular inhibitor of the urokinase-type plasminogenactivator/tissue-type plasminogen activator/plasmin fibrinolyticsystem, plays a prominent role in wound healing. However, undercertain circumstances (eg, diabetes), excessive PAI-1 expressioncontributes to induced fibrogenesis by suppressing cellular proteo-lytic activity and increasing ECM protein stability.30 Takentogether, elevated TGF-b1 and PAI-1 expression promotes exces-sive deposition of collagen and ECM proteins in cavernous tissue.However, both in vivo and in vitro studies have demonstrated thattaurine can inhibit expression of TGF-b1.31e32 In addition, Leeet al reported that taurine significantly reduced PAI-1 expression inrats with streptozotocin-induced diabetes at themRNA and proteinlevel.33 Here, taurine administration reversed the elevated expres-sion of TGF-b1 and PAI-1. As a result, collagens and ECM pro-teins, including collagen I and collagen IV, were reduced.

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Figure 4. Semiquantitative analyses of smooth muscle and collagen ratio and a-smooth muscle actin (a-SMA) expression in all groups.(A) Masson trichrome and immunohistochemical staining with antibody to a-SMA (magnification �100). (B) Representative Western blotanalysis of a-SMA expression. (C) Data are expressed as a ratio of smooth muscle to collagen. (D) Quantitative analysis of a-SMA-positivearea in cavernous tissue was performed with an image analyzer. (E) Data are presented as the relative density values of a-SMA to b-actinloading control. The relative ratio measures in the control group is arbitrarily presented as 1. *P < .05 compared with the control group.†P < .05 compared with the DED þ Taurine group. Figure 4 is available in color at www.jsm.jsexmed.org.

Taurine Improves Diabetic Erectile Dysfunction via Anti-fibrosis 783

Cavernous smooth muscle cells compose the predominantstructure of erectile tissue and are responsible for blood flowcontrol into the corpus cavernosum. In patients with DED, lossof smooth muscle cells further aggravates penile fibrosis.Increased apoptosis and reduced a-SMA expression qualifies thisview. Moreover, insulted smooth muscle cells may serve asfibrogenic effector cells, which in return, produce a variety ofmediators, cytokines, and other factors involved in inflamma-tion.24 Preservation of taurine for smooth muscle cells can breakthis vicious circle. In summary, taurine, as an antifibrosis agent,not only reduced ECM protein deposition but also protectedfunctional cells from apoptosis.

As DED is closely related to hypogonadism,34 we determinedplasma testosterone levels. In line with the results of previousstudies,35 DED rats had a marked decrease in plasma testosteronelevels. Moreover, taurine affects hormonal parameters in agedrats.11 In the present study, the levels of testosterone were increasedin DED rats treated with taurine but were still lower compared tothose of normal controls. Accordingly, the increase in plasmatestosterone levelsmight have an indirect effect on erectile function.

Our previous work (unpublished, doctorate dissertation ofDr Jun Yang) noted that APO-positive rats had only mild tomoderate ED, whereas APO-negative rats’ sexual function was

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impaired severely and often had a poorer response to PDE5 in-hibitors. In this study, we used an APO-negative model to mimicsevere ED. Our results indicate that taurine could at leastpartially ameliorate ED in APO-negative rats. This findingprovides evidence for a potential novel therapy for severe ED.Future studies are needed to validate the combination of PDE5inhibitors and taurine, which could improve the response rate innonresponders to PDE5 inhibitors.

A major limitation of the current research is that ourstreptozotocin-induced diabetes model reflects type 1 diabetesbut may not necessarily represent the pathologic processes thatoccur in the more common type of diabetes. Furthermore, wedid not thoroughly investigate the mechanisms involved in thebenefits of taurine for DED. Therefore, future studies shouldexamine the effect of taurine in vitro and determine the corre-lation with erectile function and penile fibrosis during the courseof diabetes.

CONCLUSION

Data from the present study suggest that taurine supplemen-tation may improve erectile function in rats with DED andameliorate penile fibrosis as well as endothelial dysfunction. This

784 Ruan et al

finding provides evidence that taurine supplementation may bean alternative therapy for nonresponders to PDE5 inhibitors.

Corresponding Authors: Mingchao Li, MD, PhD, Depart-ment of Urology, Tongji Hospital, Tongji Medical College,Huazhong University of Science and Technology, Wuhan430030, Hubei, China. Tel: (86) 027-83663673; Fax: (86) 02783663673; E-mail: chaoren149@163.com

Jihong Liu, MD, PhD, Department of Urology, Tongji Hospi-tal, Tongji Medical College, Huazhong University of Science andTechnology, Wuhan 430030, Hubei, China. Tel: (86) 027-83663673; Fax: (86) 027 83663673; jhliu@tjh.tjmu.edu.cn

Conflict of interest: The authors report no conflicts of interest.

Funding: This work was supported by a grant from the NationalNatural Sciences Foundation of China (No. 81300477).

STATEMENT OF AUTHORSHIP

Category 1

(a) Conception and Design

Yajun Ruan; Mingchao Li; Tao Wang; Jihong Liu

(b) Acquisition of Data

Yajun Ruan; Mingchao Li; Jun Yang; Ke Rao

(c) Analysis and Interpretation of Data

Yajun Ruan; Mingchao Li; Shaogang Wang; Weiming Yang

Category 2

(a) Drafting the Article

Yajun Ruan; Mingchao Li

(b) Revising It for Intellectual Content

Yajun Ruan; Mingchao Li; Tao Wang; Jihong Liu; Zhangqun Ye

Category 3

(a) Final Approval of the Completed Article

Yajun Ruan; Mingchao Li; Tao Wang; Jun Yang; Ke Rao;Shaogang Wang; Weiming Yang; Jihong Liu; Zhangqun Ye

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SUPPLEMENTARY DATA

Supplementary data related to this article can be found athttp://dx.doi.org/10.1016/j.jsxm.2016.02.164.