- Open Access
SjCa8, a calcium-binding protein from Schistosoma japonicum, inhibits cell migration and suppresses nitric oxide release of RAW264.7 macrophages
- Ji Liu†1, 2,
- Tong Pan†1, 2,
- Xu You†1, 2,
- Yiyue Xu1, 2,
- Jinyi Liang1, 2,
- Yanin Limpanont3,
- Xi Sun1, 2,
- Kamolnetr Okanurak3,
- Huanqin Zheng1, 2,
- Zhongdao Wu1, 2 and
- Zhiyue Lv†1, 2Email author
© Liu et al. 2015
- Received: 2 September 2015
- Accepted: 28 September 2015
- Published: 7 October 2015
Schistosomiasis is considered second only to malaria as the most devastating parasitic disease in tropical countries. Schistosome cercariae invade the host by penetrating the skin and migrate though the lungs and portal circulation to their final destination in the hepatic portal system and eventually the mesenteric veins. Previous studies have shown that the cytotoxic pathways that target schistosomulum in the lung-stage involve nitric oxide (NO) produced by macrophages. By contrast, skin-stage schistosomulas can evade clearance, indicating that they might be freed from macrophage NO-mediated cytotoxicity to achieve immune evasion; however, the critical molecules and mechanisms involved remain unknown.
Recombinant SjCa8 (rSjCa8), an 8-kDa calcium-binding protein that is stage-specifically expressed in cercaria and early skin-stage schistosomulas of Schistosoma japonicum, was incubated with mouse RAW264.7 macrophages. Effects on macrophage proliferation were determined using Cell Counting Kit-8. Next, transwell assay was carried out to further investigate the role of rSjCa8 in macrophage migration. The effects of rSjCa8 on macrophage apoptosis were evaluated using confocal microscopy and flow cytometry. Additional impacts of rSjCa8 on NO release by lipopolysaccharide (LPS)-stimulated macrophages as well as the underlying mechanisms were explored using fluorescent probe, nitric oxide signaling pathway microarray, quantitative real-time PCR, mutagenesis, and neutralizing antibody approaches.
rSjCa8 exhibited a striking inhibitory effect on macrophage migration, but did not markedly increase cell proliferation or apoptosis. Additionally, rSjCa8 potently inhibited NO release by LPS-stimulated macrophages in a dose- and time-dependent manner, and the inhibitory mechanism was closely associated with intracellular Ca2+ levels, the up-regulation of catalase expression, and the down-regulation of the expression of 47 genes, including Myc, Gadd45a, Txnip, Fas, Sod2, Nos2, and Hmgb1. Vaccination with rSjCa8 increased NO concentration in the challenging skin area of infected mice and reduced the number of migrated schistosomula after skin penetration by cercariae.
Our findings indicate that SjCa8 might be a novel molecule that plays a critical role in immune evasion by S. japonicum cercaria during the process of skin penetration. The inhibitory impacts of rSjCa8 on macrophage migration and [Ca2+]i-dependent NO release suggest it might represent a novel vaccine candidate and chemotherapeutic target for the prevention and treatment of schistosomiasis.
- Schistosoma japonicum
- Immune evasion
- Calcium-binding protein
Schistosomiasis, a parasitic zoonosis caused by schistosomes that parasitize the portal and mesenteric veins of the host, remains the second most common epidemic tropical disease worldwide, as this parasite infects 200 million individuals in 73 countries in Asia (including China), Africa, and Latin America, and is responsible for ~100,000 deaths annually [1, 2]. Schistosome is a multicellular organism with a complex life cycle, in which a sophisticated immunoevasion system impedes the development of a protective immune response [3, 4]. Because praziquantel represents the only effective drug against schistosomiasis, there is a great risk of the development of drug resistance , which represents a severe challenge in the field of schistosomiasis prevention and eradication.
The cercaria stage is a transient free stage in the S. japonicum life cycle, and cercaria invasion initiates the process of infection in its definitive host. Although cercariae are relatively simple and short-lived organisms, it is well recognized that the ultraviolet-ray (UV) attenuated cercariae can induce high and stable protection against Schistosoma mansoni challenge in many animal models [6, 7]. Furthermore, in contrast with most lung-stage schistosomula that are eliminated by macrophage NO-mediated cytotoxicity [8, 9], skin-stage schistosomulas can evade clearance by immune cells by penetrating the host’s skin surface, indicating that a specific schistosome-derived molecule might exist that facilitates immunoevasion .
Our previous studies have shown that SjCa8, an 8-kDa calcium-binding protein derived from S. japonicum, is specifically expressed in cercariae and skin-stage schistosomula, but is silenced in eggs, lung-stage schistosomula, and adult worms . Additionally, previous studies have localized SjCa8 to the cercarial head gland, penetration gland, secretions, and tegument (where cercariae directly contact and interact with host cells) , which suggest that stage-specific expression of SjCa8 could play a role in the skin penetration process by cercariae, although the mechanisms involved remain unclear. Therefore, in this study, we characterized the effects of rSjCa8 on macrophage proliferation, apoptosis, migration, and NO release.
Preparation of recombinant proteins
Purified recombinant SjCa8 (rSjCa8) and Sj13 (rSj13, control protein) were prepared as described previously  and subsequent endotoxin removal from purified proteins was performed using Detoxi-Gel Endotoxin Removal Columns (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions .
To prepare recombinant mutant SjCa8 (rmuSjCa8), full-length (210 bp) muSjCa8 DNA with site-specific mutations of glutamate (E, positions 26 and 62) in the Ca2+-binding loops, which are thought to be key residues for calcium binding [11, 14], to glutamine (Q), was synthesized by Takara Bio Inc. (Dalian, China). The mutant gene was subsequently cloned, expressed, purified, and subjected to endotoxin removal following established procedures [11, 13].
The mouse RAW264.7 macrophage-like cell line was obtained from the American Type Tissue Culture Collection (Rockville, MD, USA), cultured in Dulbecco’s Modified Essential Medium (DMEM, Gibco® Life Technologies, Grand Island, NY, USA) supplemented with 10 % heat-inactivated fetal bovine serum (FBS, Gibco® Life Technologies) and penicillin (100 U/ml) plus streptomycin (100 μg/ml), and incubated at 37 °C and 5 % CO2. Cells were seeded in a T25 flask at 5 × 105 cells/ml 1 day prior to inoculation.
Cell proliferation assay
RAW264.7 cells were seeded in 96-well culture plates 1 day prior to treatment at a density of 2 × 103 cells/well in 100 μL medium, and then were treated with either phosphate buffer saline (PBS), 20 μg/ml rSj13, 5 μg/ml concanavalin-A (ConA, positive control), or rSjCa8 at different concentrations (1 and 20 μg/ml) for either 24 or 48 h. Cell viability was assessed using Cell Counting Kit-8 (CCK-8, Beyotime Biotechnology, Haimen, China) as described previously . Absorbance at 450 nm, for positive values indicate cellular proliferation, was measured using a reference wavelength of 595 nm with an automatic Microplate Absorbance Reader (Tecan, Austria).
To detect apoptosis in RAW264.7 cells after rSjCa8 treatment, we assessed the surface phosphatidylserine (PS) expression levels, which increase during apoptosis. Exposure of the phospholipid PS to the external leaflet of the plasma membrane occurs early in the process of apoptosis and can be measured by Annexin-V-FITC staining. Moreover, dead cells that reach late stages of apoptosis can be quantified by positive PI staining. To quantify apoptosis, an early Apoptosis Detection Kit (Beyotime Biotechnology) was performed. RAW264.7 cells (105 cells/well) were grown in culture medium in the presence or absence of rSjCa8 (1 or 20 μg/ml), rSj13 (20 μg/ml), or 1:2000 (V/V) Apoptosis Inducer (positive control, Beyotime Biotechnology) for 24 or 48 h. Then, cells were harvested and pelleted by centrifugation. After washing with PBS and resuspension in binding buffer (10 mM HEPES/NaOH, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) containing 1:100 (V/V) propidium iodide (PI) and Annexin V-FITC at room temperature for 30 min in the dark, cells were analyzed using either a Beckman CytoFLEX Flow Cytometer (Beckman Coulter, Brea, CA, USA), or observed using a 40× lens under an Olympus FV 500-IX81 laser-scanning confocal microscope (Olympus Microscopy, Hamburg, Germany) with an excitation wavelength of 490 nm and emission wavelengths of 520 nm for Annexin-V-FITC and 650 nm for PI. The apoptosis rate for each group was calculated according to the following formula: apoptosis rate (%) = (number of apoptotic cells in a microscopic field/total number of cells in a microscopic field) × 100.
Transwell migration assay
In vitro migration assays were performed using transwell chambers with a 10-μm thin transparent polycarbonate membrane and an 8-micron polyester filter (Corning Inc. Corning, NY, USA). RAW264.7 cells (7.5 × 103) that were pretreated for 1 h with 20 μg/ml rSj13 or 0, 1, or 20 μg/ml rSjCa8 were added to the upper compartment where 1 % FBS DMEM was added, whereas DMEM containing 10 % FBS was added to the lower compartment. After 30 h incubation, the membrane between the two compartments was removed, fixed in methanol for 10 min, and stained with Giemsa reagent. Cells on the lower surface of the filter were photographed under an Olympus BX51WI microscope (Olympus Microscopy) and counted. Five random views were photographed and quantified.
Measurement of intracellular nitric oxide production
Intracellular NO release in individual RAW264.7 cells was determined using a fluorescent dye for NO, 4-amino-5-methylamino-2ʹ,7ʹ-difluorofluorescein diacetate (DAF-FM/DA, Beyotime Biotechnology) following a previously described protocol . To assess the effect of rSjCa8 on NO production by macrophages, RAW264.7 cells were pre-stimulated with 1 μg/ml LPS for 24 h at 37 °C, and then were treated for 30 min with various doses of rSjCa8 (0, 1, and 20 μg/ml) or control protein rSj13 (20 μg/ml). To analyze the dose- and time-dependent inhibitory activity of rSjCa8 on NO production by macrophages, LPS-stimulated RAW264.7 cells were incubated with 20 μg/ml rSjCa8 for 0, 1, 10, 20, or 30 min. Cells from all groups were loaded with 5 μM DAF-FM/DA in the dark for 30 min at 37 °C, and then were rinsed three times. Thereafter, the fluorescence intensities of cells were measured using an Olympus FV 500-IX81 laser-scanning confocal microscope (Olympus, Microscopy) with excitation and emission wavelengths of 490 and 510 nm, respectively. Furthermore, dynamic changes of fluorescence intensities of NO produced by LPS-stimulated macrophages after treatment with increasing concentrations of rSjCa8 (5, 10, 20, 40, 60 and 80 μg/ml) were recorded in time-series mode at 1.7 s intervals under a confocal microscope.
Detection of intercellular Ca2+ levels
Previous studies have shown a role for thapsigargin as an endoplasmic reticulum (ER) stressor, as well as a Ca2+-pump (ATPase) inhibitor that acts on the sarcoplasmic reticulum (SR) membrane . In the ER stress response, apoptosis is induced by thapsigargin in a manner dependent upon Ca2+ outflow from the SR along with cytoplasmic calcium overload. EDTA functions as a chelator bound to Ca2+ to reduce intercellular calcium concentrations. To test for correlations between NO production and intracellular calcium concentrations ([Ca2+]i) in macrophages, we simultaneously detected intracellular NO production and [Ca2+]i of individual RAW264.7 cells after various treatments. Cells pretreated with LPS (1 μg/ml) for 24 h were divided into 6 groups: controls treated with PBS (group I), a group treated with 20 μg/ml rSjCa8 for 30 min (group II), an internal control group treated with 20 μg/ml rSj13 for 30 min (group III), a group treated with 20 μg/ml rSjCa8 + 10 mM ethylenediaminetetra-acetic acid (EDTA, a chelator of Ca2+) for 30 min (group IV), a group treated with 20 μg/ml rSjCa8 + 30 μM thapsigargin (an inhibitor of sarco-endoplasmic reticulum Ca2+-ATPases) for 30 min (group V), and a group treated with 20 μg/ml rmuSjCa8 (group VI).
Finally, cells were loaded with 2 μM Rhod-2 AM (Dojindo Kumamoto, Kumamoto, Japan; a red fluorescent probe for intercellular Ca2+) and 5 μM DAF-FM/DA (a NO indicator) in the dark for 30 min at 37 °C. After incubation, cells were imaged using an Olympus FV 500-IX81 laser-scanning confocal microscope (Olympus Microscopy). The fluorescence of Rhod-2 AM and DAF-FM/DA were excited at 577 nm and 490 nm, and emitted at 581 nm and 510 nm, respectively.
NO PCR microarray chip
RAW264.7 cells were cultured, stimulated with 1 μg/ml LPS for 24 h, and treated with either PBS or rSjCa8 (20 μg/ml) for 30 min as described above. Total RNA from cells in both groups was prepared using TRIzol® reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) and the Mouse Nitric Oxide Signaling Pathway RT2 Profiler™ PCR Array (SABiosciences/QIAGEN Company, Fredrick, MD, USA) was performed according to the manufacturer’s protocol by KangChen Bio-Tech Inc. (Shanghai, China). In the statistical analyses, gene expression differences were considered significant if they showed an expression fold-change ≥3 between two groups with a p-value <0.05.
Real-time quantitative PCR
Primers used for quantitative RT- PCR
Worm recovery and tissue sampling
To evaluate the effects of rSjCa8 on NO production and host skin penetration by S. japonicum larvae in vivo, a modified immune challenge experiment in mice was performed as described in our previous study . Briefly, 40 mice were randomly divided into four groups of 10 mice each. The immune challenge group (Freund’s adjuvant + rSjCa8/infected group), adjuvant-treated group (Freund’s adjuvant/infected group), and challenge group (infected group) were treated as described previously . The immune-challenge group of mice was injected subcutaneously with 20 μg rSjCa8 plus complete Freund’s adjuvant (Sigma) and were boosted twice with the same amount of antigen with incomplete Freund’s adjuvant (Sigma) at 2-week intervals. The adjuvant-treated group of mice was subjected to the same immunization schedule as the immune-challenge group, but PBS replaced rSjCa8. Fourteen days after the final boost, mice in these three groups were challenged percutaneously with 50 ± 2 cercariae for 20 min using the cover glass method. Another group of 10 mice (uninfected group) was subjected to the same immunization schedule as the adjuvant group, but PBS was used in place of adjuvant and without a subsequent challenge infection with cercariae.
At 6 h post-challenge infection, all vaccinated and control mice were euthanized and the shaved skin areas that were exposed to cercariae were quickly removed. The skin tissues from half of each group of mice were cut into pieces and cultured for 24 h at 37 °C, 5 % CO2 in RPMI 1640 medium containing antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) and 10 % fetal calf serum (Invitrogen). In these cultured skin tissues, the effects of rSjCa8 on penetration and migration by cercariae were evaluated based on the number of penetrated larvae, as described previously . The skin tissues from the other half of each group were cut into pieces and lysed with Lysis Buffer (Thermo Scientific/Pierce; 1 ml/g skin), and skin homogenates were prepared using a TissueLyser II (Qiagen) and centrifuged at 10,000 g for 5 min at 4 °C to collect supernatants. The Total Nitric Oxide Assay kit (Beyotime Biotechnology) was used for NO detection following the manufacturer’s protocol and NO levels in the skin homogenate supernatants were determined by measuring nitrites with Griess reagent . Absorbance was read at 540 nm with an automatic Microplate Absorbance Reader (Tecan, Austria).
Each experiment was repeated three times and data were represented as means ± standard derivation (SD). The statistical significance of differences between groups was determined using a one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test using GraphPad Prism version 5.0 (GraphPad Software Inc., La Jolla, CA, USA); P < 0.05 was used as a threshold for statistically significant differences.
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Sun Yat-sen University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Zhongshan School of Medicine, Sun Yat-sen University (Permit Number: 2010–0326). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
rSjCa8 does not affect macrophage proliferation
rSjCa8 does not induce macrophage apoptosis
rSjCa8 inhibits macrophage migration
rSjCa8 inhibits LPS-induced NO release by RAW264.7 cells
Suppression of NO release by rSjCa8 in RAW264.7 cells is correlated with changes in intracellular calcium levels
Transcriptional profiles of genes involved in the NO signaling pathway are markedly altered in LPS-stimulated macrophages treated with rSjCa8
General information of differentially expressed genes from LPS + rSjCa8 treated macrophages compared to LPS treated macrophages
(LPS + rSjCa8)/LPS
Amyotrophic lateral sclerosis 2 (juvenile) homolog (human)
Bcl2-associated X protein
Calpain, small subunit 1
Copper chaperone for superoxide dismutase
Cyclin-dependent kinase inhibitor 1A (P21)
Cytochrome b-245, alpha polypeptide
Discs, large homolog 4 (Drosophila)
Fas (TNF receptor superfamily member 6)
FBJ osteosarcoma oncogene
Growth arrest and DNA-damage-inducible 45 alpha
Glutathione peroxidase 1
Glutathione peroxidase 3
High mobility group box 1
Isocitrate dehydrogenase 1 (NADP+), soluble
Immunity-related GTPase family M member 1
Transformed mouse 3 T3 cell double minute 2
Neutrophil cytosolic factor 2
Neural precursor cell expressed, developmentally down-regulated gene 1
Nitric oxide synthase 2, inducible
NADPH oxidase 1
Phosphoprotein enriched in astrocytes 15A
Protein phosphatase 1, regulatory (inhibitor) subunit 15b
Protein phosphatase 3, catalytic subunit, alpha isoform
RecQ protein-like 4
Harvey rat sarcoma oncogene, subgroup R
Stearoyl-Coenzyme A desaturase 1
Stearoyl-Coenzyme A desaturase 2
Stearoyl-coenzyme A desaturase 3
Superoxide dismutase 1, soluble
Superoxide dismutase 2, mitochondrial
Transformation related protein 53
Thioredoxin interacting protein
Thioredoxin reductase 2
Mediator of RNA polymerase II transcription, subunit 4 homolog (yeast)
Hypoxanthine guanine phosphoribosyl transferase 1
Validation of differentially expressed genes detected by microarray analyses
Vaccination of rSjCa8 increase NO concentration in the challenging skin area and reduce the number of larvae that penetrate and migrate through the skin
Ca2+ and NO are two important cellular messengers, and Ca2+ fluxes have been recognized to account for NO production . Researchers have confirmed that both Ca2+ fluxes and calcium-related proteins, such as regucalcin, calmodulin, and CBPs, are thought to affect NO synthesis and release by a range of somatic cells, including renal cortical cells, neurons, macrophages, and plant cells [21–25]. In the innate immune response, calcium-binding proteins act as potential sensors that are activated by pathogen-induced calcium influx. Increased cytoplasmic Ca2+ levels trigger the activation of various downstream protein targets that affect numerous signal transduction cascades, including the NOS (nitric oxide synthase) pathway that has been associated with increased NO generation after pathogen infection in vivo and in vitro . However, the importance of calcium-binding proteins from pathogens (e.g., parasites, bacteria, or viruses) on macrophage release or functions in the host has not yet been directly established [27–29]. To date, SjCa8 is the only CBP known to be stage-specifically expressed in schistosomal cercaria and skin-stage schistosomula . Our findings originally indicated that rSjCa8 could significantly inhibit macrophage migration and NO release, despite its minimal impact on macrophage proliferation and apoptosis. Furthermore, our present findings show that mutations in the EF-hand motif of rSjCa8 or administration of thapsigargin (an agonist of store-operated Ca2+ channels) can reverse rSjCa8-induced inhibitory effects on NO release in LPS-stimulated macrophages demonstrated that the inhibition effects might be regulated by Ca2+-dependent signaling. We have now made the exciting observation that rSjCa8 can both modulate intracellular Ca2+ levels and also inhibit macrophage migration and NO production in the cercariae-challenged host, thereby suppressing host cell-mediated killing or elimination effects on a pathogen.
Recent work indicates that LPS, a ubiquitous component of gram-negative bacteria, can elicit innate immune responses in both animals and plants by functioning as a pathogen-associated molecular pattern (PAMP)-like molecule, which can evoke NO generation in macrophages , demonstrating a useful model of RAW264.7 macrophages upon LPS stimulation for investigating pathogen-mediated NO signaling cascades. Based on our microarray NO chip analysis and quantitative real-time PCR results, we can conclude that the mechanisms of inhibiting NO release by rSjCa8 may include up-regulated expression of Catalase-encoding gene (Cat) and down-regulated expression of 47 other genes that include Myelocytomatosis oncogene (Myc), Superoxide dismutase 2 (Sod2), Thioredoxin interacting protein (Txnip), Growth arrest and DNA-damage- inducible 45 alpha (Gadd45a), TNF receptor superfamily member 6 (Fas), High mobility group box 1 (Hmgb1), and Nitric oxide synthase 2 (Nos2), which have been established to be involved in NO production and biological functions [31–40]. Since LPS-stimulated iNOS activation in RAW 264.7 macrophages is reactive oxygen species (ROS)-dependent [41–43], the observation that extremely high expression level of Cat (an important molecule in protecting the cell from oxidative damage by ROS) in LPS-pretreated macrophages is induced by rSjCa8 suggests ROS are involved in the inhibitory effect of rSjCa8 on NO release. LPS-stimulated macrophages are a critical component of innate immunity. After LPS treatment, protein-tyrosine kinase mediates phospholipase C phosphorylation, which is followed by increased intracellular calcium concentrations. The increases in calcium levels sequentially trigger the activation of downstream signaling pathways that lead to inducible nitric-oxide synthase [44, 45]. Our findings presented herein clearly show that although rSjCa8 clearly inhibits Toll-like receptor (TLR) activation-induced (e.g., LPS exposure) NO production, non-TLR agonists (e.g., thapsigargin exposure) can abolish the reduction in NO and intracellular Ca2+ accumulation in LPS-stimulated macrophages. Thus, inhibition of Ca2+-mediated inflammatory pathways likely represents a novel schistosome-induced immunosuppressive mechanism. Deeper insights into the molecules and mechanisms related to the induction of NO production by rSjCa8 both permit a more thorough characterization of the NO signaling system and lead to a better understanding of immune evasion by S. japonicum.
Increasing studies have confirmed the existence of cross-talk between Ca2+ signaling and NO in response to pathogen invasion [46–48]. To further test our hypothesis that immune evasion of cercariae during skin penetration was mediated by the inhibitory activity of SjCa8 on NO production, we immunized mice with adjuvant + rSjCa8 to induce significantly higher levels of specific anti-rSjCa8 antibodies compared to those in mice immunized with adjuvant or PBS alone (data not shown). Our findings indicate that the inhibitory activity of rSjCa8 on NO production can be suppressed by neutralizing antibodies to rSjCa8. Therefore, accumulation of NO in the challenging skin area of infected hosts caused the partial elimination of invading larvae, which can further illustrate why schistosomal cercariae are scarcely killed or eliminated while penetrating into the host’s skin. Cercariae and skin-stage schistosomula might evade the host immune system-mediated elimination by secreting SjCa8, a stage-specific CBP, which can inhibit macrophage migration and NO release. Therefore, SjCa8 is thought to be a potential chemotherapeutic target or/and vaccine candidate against schistosome infection.
Our findings support the conclusion that SjCa8, a cercaria and skin-stage schistosomulum specific CBP, inhibits cell migration and Ca2+-dependently suppresses NO release by LPS-stimulated RAW264.7 macrophages. It is feasible that, via this mechanism, SjCa8 can contribute to preventing larvae from the damaging or killing effects of macrophages, thus suggesting a further protective effect of this immunoregulatory molecule against host immune attack to promote the invasion, migration, and survival of cercariae. Our findings provide molecular insights that could be harnessed to develop a novel effective vaccine or drug against cercariae invasion for the prevention and control of schisotosomiasis.
We thank Mrs. Guan Yuanjun and Mrs. Jie Liu for providing help with confocal microscopy and expert technical assistance. This work was supported by grants from the National Natural Science Foundation of China (grant no. 30800966, 81572014, 81371836 and 81572023), the Laboratory of Parasite and Vector Biology, MOPH (grant no. WSBKTKT201401), the Guangdong Natural Science Foundation (grant no. 2014A030313134), the 111 Project (grant no. B12003), the Cultivation Foundation of the Young Teacher of Sun Yat-sen University (grant no. 09ykpy77), and the Research Foundation for Students of Sun Yat-sen University (2012 and 2014).
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