Rab27a/Slp2-a complex is involved in Schwann cell myelination

Wen-feng Su, Yun Gu, Zhong-ya Wei, Yun-tian Shen, Zi-han Jin, Ying Yuan,, Xiao-song Gu, Gang Chen, Jiangsu Key Laboratory of Neuroregeneration, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, China Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China

Rab27a/Slp2-a complex is involved in Schwann cell myelination

Wen-feng Su1, Yun Gu1, Zhong-ya Wei1, Yun-tian Shen1, Zi-han Jin1, Ying Yuan1,2, Xiao-song Gu1, Gang Chen1,*
1 Jiangsu Key Laboratory of Neuroregeneration, Co-innovation Center of Neuroregeneration, Nantong University, Nantong, Jiangsu Province, China
2 Affiliated Hospital of Nantong University, Nantong, Jiangsu Province, China

How to cite this article:Su WF, Gu Y, Wei ZY, Shen YT, Jin ZH, Yuan Y, Gu XS, Chen G (2016) Rab27a/Slp2-a complex is involved in Schwann cell myelination. Neural Regen Res 11(11):1830-1838.

Open access statement:This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.

Funding:This study was supported by the National Natural Science Foundation of China, No. 31071251, 81471255, and 81471259; a grant from the Ministry of Science and Technology of China (973 Program), No. 2014CB542202; a grant from the Basic Research Program of Education Department of Jiangsu Province, China, No. 14KJA310004; a grant from the Natural Science Research Project of Nantong Science and Technology Bureau, China, No. HS2013014; and a grant from the Natural Science Research Project of Nantong University, China, No. 13Z008.

Graphical Abstract

Myelination of Schwann cells in the peripheral nervous system is an intricate process involving myelin protein trafficking. Recently, the role and mechanism of the endosomal/lysosomal system in myelin formation were emphasized. Our previous results demonstrated that a small GTPase Rab27a regulates lysosomal exocytosis and myelin protein trafficking in Schwann cells. In this present study, we established a dorsal root ganglion (DRG) neuron and Schwann cell co-culture model to identify the signals associated with Rab27a during myelination. First, Slp2-a, as the Rab27a effector, was endogenously expressed in Schwann cells. Second, Rab27a expression significantly increased during Schwann cell myelination. Finally, Rab27a and Slp2-a silencing in Schwann cells not only reduced myelin protein expression, but also impaired formation of myelin-like membranes in DRG neuron and Schwann cell co-cultures. Our findings suggest that the Rab27a/ Slp2-a complex affects Schwann cell myelinationin vitro.

nerve regeneration; Schwann cells; dorsal root ganglion neurons; co-culture; myelin proteins; myelination; Rab27 effectors; Rab27a; Slp2-a; neural regeneration

Introduction

The myelin sheath in the peripheral nervous system (PNS) comprises a specialized Schwann cell plasma membrane, which serves to increase axonal impulse conduction (da Silva et al., 2014; Luo et al., 2014; Nave and Werner, 2014; Miyamoto et al., 2016; Taveggia, 2016). Clinically, abnormal myelination is a feature of many peripheral neuropathies that can cause abnormal electrical signal conduction and lead to secondary axonal injury (Duncan et al., 2014; Gonzalez et al., 2016; Klein and Martini, 2016; Kondo and Duncan, 2016; Schulz et al., 2016). The proper synthesis and transport of myelin proteins is important for myelin biogenesis (Kwon et al., 2013; Heller et al., 2014; Montani et al., 2014; Domènech-Estévez et al., 2015; G?kbuget et al., 2015).However, the mechanisms regulating myelin protein trafficking remain poorly understood (White and Kr?mer-Albers, 2014; Salzer, 2015; Ma et al., 2016; Rao and Pearse, 2016).

Recently, the role and mechanisms of the endosomal/lysosomal system in myelin formation were investigated (Trajkovic et al., 2006; Prolo et al., 2009; Feldmann et al., 2011; Shen et al., 2016). Neuronal signals induce exocytose is of the proteolipid protein (PLP) from late endosome/lysosome membranes stored into the plasma membrane (Trajkovic et al., 2006; Shen et al., 2016). Consistent with this finding, myelin abnormalities are a very common phenomenon in many lysosomal storage diseases, including multiple sulfatase deficiency, globoid cell leukodystrophy, and metachromatic leukodystrophy (Faust et al., 2010; Marsden and Levy, 2010). Because lysosomes serve dual functions in many cell types–the degradation of proteins and storage of synthesized secretory products (Blott and Griffiths, 2002; de Duve, 2005; Zhang et al., 2007; Johnson et al., 2013; Kim et al., 2013; Hou et al., 2015; Shimada-Sugawara et al., 2015)–the concept of secretory lysosomes was proposed.

Previous studies have shown that the Rab27 subfamily and their multiple effectors play a critical role in regulating lysosome-related organelle exocytosis (Izumi, 2007; Johnson et al., 2013; Shimada-Sugawara et al., 2015; Yamaoka et al., 2015b, a). Additionally, our previous results showed that Rab27a participates in lysosomal exocytosis and myelin protein P0 trafficking in Schwann cells. Furthermore, Rab27a-deficient ashen mice demonstrate impaired demyelination of the injured sciatic nerve (Chen et al., 2012). In this study, we further investigated the mechanisms of Rab27a in the regulation of Schwann cell myelination by the dorsal root ganglion (DRG) neuron and Schwann cell co-culture system as a model of myelination.

Material and Methods

Culture, isolation, and purification of Schwann cells

Animal protocols were reviewed and approved by the Animal Ethics Committee of Nantong University, China (license No. 2014-0001), and the experimental protocol was in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication, No. 80-23). Precautions were taken to minimize suffering and the number of animals used in each experiment.

Primary cultures of Schwann cells were prepared as previously described (Brockes et al., 1981; Chen et al., 2012). In brief, Schwann cells were harvested from sciatic nerves of 1–3-day-old Sprague-Dawley rats (Experimental Animal Center of Nantong University, Nantong, Jiangsu Province, China, SPF level, no gender requirement). To inhibit fast proliferation of fibroblasts, the Schwann cell purification medium was replaced (Table 1) for 1 day, followed by a 1-day recovery period in growth factor-free medium, then refreshed with Schwann cell growth medium. About 7 days later, when the Schwann cell cultures reached confluence, complement-mediated immune cytolysis was performed to eliminate fibroblasts by incubating the cells in 4 μg/mL of anti-Thy-1.1 antibody (Sigma-Aldrich, St. Louis, MO, USA) for 2 hours on ice and then with 1 mL of rabbit complement (Gibco, Carlsbad, CA, USA) for 1 hour at 37°C. Afterwards, > 98% pure Schwann cells were obtained and the purity was determined by immunocytochemistry.

Culture, isolation, and purification of DRG neurons

DRG neurons were isolated by dissection from embryos of 14.5-day pregnant Sprague-Dawley rats (Experimental Animal Center of Nantong University, Nantong, Jiangsu Province, China. SPF level). One DRG explant was placed into each poly-D-lysine (PDL) (Invitrogen)-coated tissue culture well (24-well dishes, glass cover slips covered with 10 μg/mL PDL before use) along with high-glucose Dulbecco’s-modified Eagle’s medium (DMEM-HG) (Gibco) containing 10% fetal bovine serum (FBS) (Gibco) and the cells were allowed to adhere for 24 hours. The medium was then replaced with DRG purification medium (Table 1) and the non-neuronal cells were eliminated by incubating for 3 days in DRG purification medium. The medium was then replaced with DRG growth medium followed by the addition of purified Schwann cells.

Co-culture of DRG neurons and Schwann cells

The DRG neuron and Schwann cell co-culture system was prepared as previously described (Eldridge et al., 1989). Briefly, purified Schwann cells were co-cultured and growth factors were removed at 4 hours prior to plating on DRG neurons. Then, 0.125% trypsin was used to digest Schwann cells into single cells, and 50,000 Schwann cells were added to each DRG neuron culture coverslip. DRG growth medium was changed 1 day before co-culture, and half of the original medium was discarded after co-culture. A total of 250 μL DMEM-HG containing 10% FBS was added to each well, and the cells were allowed to attach overnight. The DRG neuron and Schwann cell co-cultures were maintained in DRG growth medium for 2 days and then switched to differentiation medium for 4 days. Finally, the co-culture system was maintained in myelination medium to induce myelination. Half of the medium volume was changed every 2 days. After 3–4 days, myelin sheaths were present. In this co-culture process, several specific myelination stages were identified by immunocytochemistry and electron microscopy.

siRNA transfection

Schwann cells were transfected with siRNA using Lipofectamine? RNAi MAX complexes (Invitrogen) at 4 or 5 days post-purification. ON-TARGET plus Non-targeting control siRNA (catalog D-0018100-01-20, GE Dharmacon, Lafayette, CO, USA) served as the control. The siRNA primer sequences for Rab27a and siRNA Slp2-a are listed inTable 2.

Real-time quantitative PCR assay

Total RNAs were extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany) and cDNA was synthesized using a cDNA Reverse Transcription Kit (Applied Biosystems, Foster, CA, USA). Real-time quantitative PCR was performed using the 7300 Real-Time PCR System (Applied Biosystems). The primers used in this experiment are shown inTable 3.Levels of mRNA were normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a reference. All reactions were repeated in triplicate.

Immunocytochemistry

The culture medium was aspirated, and the cells were washed with PBS and then fixed with 4% paraformaldehyde (PFA) for 15 minutes at room temperature and methanol for 10 minutes at ?20°C. The non-specific antibody-binding sites were blocked with Immunol Staining Blocking Buffer (Beyotime, Haimen, Jiangsu Province, China) for 1 hour at room temperature, and then the cultures were incubated with primary antibody overnight at 4°C. Antibodies to the following proteins were used at the indicated concentrations: mouse anti-Rab27a (1:100; Abcam, Cambridge, MA, USA), rabbitanti-Slp2-a (1:200; Protein Tech, Chicago, IL, USA), mouse anti-MBP (1:200, Abcam), chicken anti-P0 (1:50; Novus, Littleton, CO, USA), rabbit anti-MAG (1:200; Sigma), and mouse anti-NF (1:600; Sigma). After washing 3 times in PBS to remove excess primary antibody, the cultures were incubated for 1 hour at room temperature with their respective fluorescence-conjugated secondary antibodies (Jackson Immuno Research, West Grove, PA, USA): goat anti-rabbit IgG-Cy3 (H + L), goat anti-mouse IgG-Cy3 (H + L), goat anti-rabbit IgG-Cy5 (H + L), goat anti-mouse IgG-Alexa-488 (H + L), goat anti-rabbit IgG-Alexa-488 (H + L), and goat anti-chicken IgG (H + L) rhodamine, all at a dilution of 1:1,000. After a final PBS washing, the stainings were imaged with a confocal microscope (TCS SP5; Leica Microsystems, Wetzlar, Germany).

Electron microscopy

The co-cultures were examined using electron microscopy at different stages as described (Einheber et al., 1995; Yuan et al., 2004). At the exact stage, the co-culture coverslips were fixed with precooled 2.5% glutaraldehyde (Sigma) and preserved at 4°C.

Scanning electron microscopy

The co-culture cells were washed with tannic acid and then post-fixed with 1% osmium tetraoxide solution (Sigma), dehydrated stepwise twice in increasing concentrations of ethanol and in Tert-butanol, and dried in a critical point drier (Hitachi, Tokyo, Japan). Subsequently, the samples were coated with gold in a JFC-1100 unit (Jeol Inc., Tokyo, Japan) and observed under a scanning electron microscope (JEM-T300, Jeol Inc.).

Transmission electron microscopy

The co-culture coverslips were embedded in 1% agar suitable for obtaining cross-sections of the cultures, post-fixed with 1% osmium tetraoxide solution (Sigma), dehydrated stepwise in increasing concentrations of ethanol, embedded in Epon 812 epoxy resin (Sigma), and cut into transverse sections. Then the ultra-thin transverse sections were stained with lead citrate and uranyl acetate. These sections were examined under a transmission electron microscope (Jeol Inc.).

Western blot assay

Protein was extracted from cultured cells with a buffer containing 1% sodium dodecyl sulfate (SDS, Sigma), 100 mM Tris-HCl, 1 mM phenylmethylsulfonyl fluoride (PMSF, Sigma), and 0.1 mM β-mercaptoethanol (Sigma). After centrifugation at 12,000 r/min for 5 minutes, the supernatant was collected and the protein concentration was determined. Protein extracts were heat denatured at 100°C for 5 minutes, electrophoretically separated on 8% or 12% SDS-PAGE, and transferred to polyvinylidene fluoride (PVDF) membranes (Thermo Fisher Scientific Pierce, Walthan, MA, USA). The membrane was probed with antibodies specific to Rab27a (mouse, 1,000, abcam), Slac2-b (goat, 1:200, SANTA CRUZ Inc., Paso Robles, CA, USA), Slac2-c (rabbit, 1:200, SANTA CRUZ Inc.), Slp2-a (rabbit, 1:800, Protein tech Group, Inc., Chicago, IL, USA), Slp3-a (rabbit, 1:200, Santa Cruz Inc.), Slp4 (rabbit, 1:500, Anbo, San Francisco, CA, USA), MAG (rabbit, 1:800, Sigma), P0 (chicken, 1:800, Novus, Littleton, CO, USA), and PMP22 (rabbit, 1:800, abcam) overnight at 4°C, followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1,000, Beyotime) for 1 hour at room temperature, and detected using the enhanced chemiluminescent substrate kit (ECL, Thermo Fisher Scientific Pierce, Walthan, MA, USA). The image was scanned with a GS800 Densitometer Scanner (Bio-Rad), and the data were analyzed using PDQuest 7.2.0 software (Bio-Rad). β-Actin (mouse, 1:5,000, Sigma) was used as an internal control (Gu et al., 2012; Wang et al., 2012).

Immunoprecipitation

Cultured Schwann cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Millipore, Billerica, MA, USA) and the lysates were incubated with 10 μg/mL mouse anti-Rab27a antibody (abcam) or mouse IgG control (abcam) on ice overnight with occasional shaking. The antibody-protein complexes were aggregated with Protein G-Agarose (Pierce) overnight on ice, with occasional shaking, and then centrifuged at 8,000 ×gfor 10 minutes. The pellet was washed for elimination of non-specific binding with 1 × RIPA buffer, eluted by 4 × SDS sample buffer without DL-dithiothreitol (DTT), boiled for 10 minutes, and then processed for western blotting.

Statistical analysis

All data are expressed as the mean ± SEM. Differences between two groups were compared using the Student’st-test. One-way analysis of variance followed by the Newman-Keuls test was used for the statistical analyses in other tests. The criterion for statistical significance wasP< 0.05.

Results

Identification of Slp2-a as Rab27a effector in Schwann cells

Complement-mediated cytolysis with anti-Thy-1.1 antibody was used to purify cultured primary Schwann cells to remove contaminating of fibroblasts. To investigate endogenous expression of Rab27 protein family members in Schwann cells, real time-PCR and double staining were used to detect purified Schwann cells. As shown inFigure 1A–C,Schwann cells only expressed Rab27a protein, although real time-PCR results also showed weak Rab27b expression. Results are consistent with previous reports that Rab27a is primarily expressed outside the CNS and Rab27b is expressed in the central nervous system (Izumi, 2007). To identify the Rab27a effectors in Schwann cells, we analyzed mRNA expression of 11 different Rab27a/b effectors in Schwann cells according to a previous study (Izumi, 2007). As shown inFigure 1D, expressions of Slac2-b, Slac2-c, Slp2-a, Slp3-a, and Slp4 mRNA were readily visualized in Schwann cells. Western blotting identified high expression of only Slp2-a in Schwann cells (Figure 1E). Double Slp2-a and Rab27a immunostaining revealed co-localization of Slp2-a with Rab27a in cultured Schwann cells (Figure 1F). Additionally, co-immunoprecipitation experiments clearly showed an interaction between Slp2-a and Rab27a in cultured Schwann cells (Figure 1G). These results suggested that Slp2-a is endogenously expressed as a Rab27a effector in Schwann cells.

Table 1 Different culture media for growth, purification, differentiation, and myelination of Schwann cells and dorsal root ganglion (DRG) neurons

Table 3 Real time-PCR primers used for Rab27a/b effectors

Table 2 siRNA primer sequences for Rab27a and siRNA Slp2-a

Myelination model of DRG neurons co-cultured with Schwann cells

Figure 1 Identification of Slp2-a as a Rab27a effector in cultured Schwann cells.

Figure 2 Establishing a myelination model of DRG neurons co-cultured with Schwann cells.

Figure 3 DRG neurons co-cultured with Schwann cells induce persistent up-regulation of Rab27a in Schwann cells.

Figure 4 Rab27a and Slp2-a siRNA reduces Rab27a and Slp2-a expressions in cultured Schwann cells.

Figure 5 Knockdown of Rab27a and Slp2-a in Schwann cells reduces myelin protein expression and myelin-like membrane formation in the DRG neuron and Schwann cell co-cultures.

To examine the role of Rab27a and Slp2-a in Schwann cells on myelin formationin vitro, we established a myelination model of DRG neurons co-cultured with Schwann cells. Previous groups have successfully used this model to study PNS myelination (Johnson et al., 2001; Liu et al., 2005; Court et al., 2011). As shown inFigure 2, about 2 weeks after Schwann cells were added to the pure DRG neuron cultures, phase-contrast and fluorescence microscopy revealed myelin sheaths in the co-culture. Myelin sheaths were identifiable by the segregation structure in compact myelin sheaths under phase-contrast microscopy observation (Figure 2A) and the segregation of MAG-positive signalsby immunostaining (Figure 2B). Scanning electron microscopy (Figure 2C) revealed that Schwann cells surrounded the DRG neuronneurites and formed myelin segments, with a diameter longer than that of unmyelinated neurites. Transmission electron microscopy also showed the typical compact myelin structure (Figure 2D). Myelin protein expressions, including MAG, P0, and PMP22, were detected by western blotting at 1, 3, 7, 14, 21, 28, and 35 days after co-culture. Compared with purified Schwann cells, the DRG neuron and Schwann cell co-culture resulted in increased expression of all myelin proteins, which peaked at 35 days (Figure 2E). These results showed that a stable and reliablein vitromyelination model was established.

Silencing of Rab27a and Slp2-a in the DRG neuron and Schwann cell co-culture reduces myelin protein expression and impairs formation of myelin-like membranes

Using the DRG neuron and Schwann cell co-culture myelination model, we examined expression of Rab27a and Slp2-a at different time points following co-culture. As shown inFigure 3, western blot results showed significantly increased Rab27a expression at 3 days, which peaked at 7 days and remained increased at 13 days. However, Slp2-a expression remained unchanged after co-culture.

Next, siRNA silencing of Rab27a and Slp2-a was used in Schwann cells to examine the roles of Rab27a and Slp2-a in myelination. First, the gene silencing efficiency of siRNA was detected using the same sequence of siRNA-Rab27a as our previous report (Chen et al., 2012), as well as three different siRNA-Slp2-a sequences. Gene silencing efficiency was then compared by real-time quantitative PCR. As shown inFigure 4A–C, siRNA dramatically reduced Rab27a and Slp2-a levels in Schwann cells. Among the three different sequences of Slp2-a siRNA, sequence #3 exhibited the strongest effect. Expressions of Rab27a and Slp2-a protein were analyzed in Schwann cells by western blotting. As shown inFigure 4D–F, Rab27a and Slp2-a protein expressions were dramatically reduced.

Finally, purified Schwann cells were transfected with siRNA prior to seeding with DRG neurons, and then myelin protein mRNA levels and protein expressions were determined. As shown inFigure 5A–D, compared with the control group, Rab27a and Slp2-a knockdown significantly reduced both mRNA levels and protein expression of MAG, P0, and PMP22 after 14 days in co-culture. Furthermore, MBP immunostaining after 14 days of co-culture also showed that Rab27a or Slp2-a silencing impaired the formation of myelin-like membranes (Figure 5E,F). These results suggest that the Rab27a-Slp2-a complex plays an important role in Schwann cell myelination, especially in the early stage.

Discussion

The onset and maintenance of myelination in the PNS depends on myelin protein synthesis and transport (Salzer, 2015; Kondo and Duncan, 2016; Schulz et al., 2016; Taveggia, 2016). Our previous results indicate that Rab27a regulates lysosomal exocytosis and is involved in myelin protein trafficking in Schwann cells (Chen et al., 2012). This study aimed to identify the signals associated with Rab27a in Schwann cells. Our results demonstrated the importance of the Rab27a/Slp2-a complex in Schwann cell myelination. The underlying mechanism was determined through multiple experimental approaches, showing 1) Slp2-a was endogenously expressed as a Rab27a effector in Schwann cells; 2) thein vitroSchwann cell myelination model was established in our experiment; 3) Rab27a expression was significantly increased during Schwann cell myelination; 4) silencing of Rab27a and Slp2-a in Schwann cells not only reduced myelin protein expression, but also impaired the formation of myelin-like membranes in DRG neuron and Schwann cell co-cultures.

Rab27a is associated with secretory lysosomes and intracellular protein trafficking (Blott and Griffiths, 2002; Izumi, 2007; Johnson et al., 2013; Catz, 2014; Ishida et al., 2014; Chen et al., 2015; Feng et al., 2016). It has been proposed that Rab27a regulates different membrane transport events by interacting with a different set of specific effectors (Li et al., 2014; Yasuda and Fukuda, 2014; Yamaoka et al., 2015a, 2016; Yasuda et al., 2015; Jiang et al., 2016; Kowluru, 2016; Netter et al., 2016). Three groups of Rab27a effectors from a total of 11 Rab27a/b effectors, including synaptotagmin-like protein (Slp), Slp homologue lacking C2 domains (Slac2), and Munc13-4, have been identified in mice and humans (Fukuda, 2005, 2013; Izumi, 2007; Ishida et al., 2014; Jiang et al., 2016; Kowluru, 2016; Netter et al., 2016). Results from the present study showed that Slp2-a and its binding effector Rab27a form a complex in Schwann cells. In general, Slp2-a can promote docking of Rab27a-containing vesicles to the plasma membrane in certain types of secretory cells (Kuroda and Fukuda, 2004; Saegusa et al., 2006; Holt et al., 2008; Ménasché et al., 2008; Fukuda, 2013; Yasuda and Fukuda, 2014; Yasuda et al., 2015). Although the Rab27a effector function of Slp2-a has been well established, the role of Slp2-a in myelination remains poorly understood. Results from the present study showed that Slp2-a silencing in Schwann cells not only reduced myelin protein expression, but also impaired formation of myelin-like membranes in the DRG neuron and Schwann cell co-cultures. The mechanisms responsible for decreased myelin protein expression by Rab27a and Slp2-a knockdown are not clear, but could be due to negative feedback of myelination.

Thein vitromodel of rat DRG explant and Schwann cell co-cultures has been used in various studies (Johnson et al., 2001; Liu et al., 2005; Court et al., 2011; Liu and Chan, 2016) to determine the intrinsic molecular mechanisms responsible for myelination, including the complex regulation of transcription factors and myelin protein during myelin formation (Pereira et al., 2012; Salzer, 2015). In this study, we established a stable and reliablein vitromyelination model and transmission electron microscopy images confirmed the typical compact myelin structure. We also analyzed expression of several myelin proteins in this model using western blot analysis at different time points.

Above all, this is the first demonstration that the Rab27a/Slp2-a complex affects Schwann cell myelinationin vitro. These findings show an interesting link between myelin biogenesis and lysosome-related organelle exocytosis, providing a basis for clinical treatments of demyelinating diseases.

Acknowledgments:We are very grateful to Mr. Yul Huh from the Duke-NUS Medical School for instructive comments on the paper.

Author contributions:WFS conceived the study, performed the experiments, and analyzed the data. YG designed the experiment; ZYW, YTS and ZHJ performed the experiment. GC, YG, YY and XSG formulated the hypotheses and reviewed the paper. GC and WFS wrote the paper. All authors approved the final version of the paper.

Conflicts of interest:None declared.

Plagiarism check:This paper was screened twice using CrossCheck to verify originality before publication.

Peer review:This paper was double-blinded and stringently reviewed by international expert reviewers.

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Copyedited by Copper C, Norman C, Su WF, Yu J, Li CH, Li JY, Song LP, Zhao M

*Correspondence to: Gang Chen, Ph.D., M.D., chengang6626@ntu.edu.cn.

orcid: 0000-0003-3669-5687 (Gang Chen)

10.4103/1673-5374.194755

Accepted: 2016-10-19