Oral administration of recombinant human granulocyte- macrophage ...

Biotechnol Lett (2008) 30:1679–1686 DOI 10.1007/s10529-008-9717-2

ORIGINAL RESEARCH PAPER

Oral administration of recombinant human granulocytemacrophage colony stimulating factor expressed in rice endosperm can increase leukocytes in mice Tingting Ning Æ Tingting Xie Æ Qingchuan Qiu Æ Wei Yang Æ Shunquan Zhou Æ Limei Zhou Æ Congyi Zheng Æ Yingguo Zhu Æ Daichang Yang

Received: 13 March 2008 / Revised: 24 March 2008 / Accepted: 31 March 2008 / Published online: 19 April 2008 Ó Springer Science+Business Media B.V. 2008

Abstract Human granulocyte-macrophage colony stimulating factor (hGM-CSF) is used clinically to treat leucopenia typically caused by cancer chemotherapy or radiotherapy. This study used multiple strategies to obtain very high expression levels of OsrhGM-CSF (14 lg/seed) in rice endosperm. Electron micrographs of immunogold-labeled transgenic endosperm showed that rhGM-CSF was not only localized in protein bodies but was also distributed in the apoplast. A biological activity assay indicated that OsrhGM-CSF stimulated the growth of TF-1 cells in vitro. In addition, the transgene was used to effectively treat leucopenia by oral administration of the unprocessed transgenic grains. In cyclophosphamide-induced leucopenic mice, transgenic seeds produced a 27% (t = 0.021) gain in leukocytes after 14 days feeding. Even in non-leucopenic mice, leukocyte gain was 37% (t = 0.002) more than that T. Ning T. Xie Q. Qiu Y. Zhu D. Yang (&) Engineering Research Center for Plant Biotechnology and Germplasm Utilization, Ministry of Education, Department of Genetics, College of Life Sciences, Wuhan University, Wuhan 430072, P.R. China e-mail: [email protected] W. Yang C. Zheng State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan 430072, P.R. China S. Zhou L. Zhou Medical School, Wuhan University, Wuhan 430072, P.R. China

of mice fed non-transgenic seeds. This study provides a novel approach to the use of oral unprocessed transgenic OsrhGM-CSF seeds to treat leucopenia. Keywords Human granulocyte-macrophage colony stimulating factor Leukocyte Oral administration Rice endosperm TF-1 Cells

Introduction Human granulocyte-macrophage colony-stimulating factor (hGM-CSF) is a glycoprotein hormone which stimulates the proliferation and differentiation of hemopoietic cells in vitro and in vivo. It is produced by multiple cell types in humans, such as fibroblasts, endothelial cells, stromal cells and lymphocytes and is widely present throughout the body in very low concentrations (Metcalf 1991). Due to its important role in the immune system, hGM-CSF is widely used to treat the symptoms of leucopenia and oral mucositis commonly caused by cancer chemotherapy or radiotherapy. In addition, it is used to facilitate the recovery of lymphocytes after bone marrow transplantation and AIDS therapy (Vazquez et al. 1998). Human GM-CSF is normally administered by intravenous injection, but recent evidence has shown that oral administration is also effective. Michael et al. (1996) studied the oral administration of recombinant hGM-CSF (rhGM-CSF) to treat radiotherapy

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(RT)-induced esophagitis and found that topical rhGM-CSF therapy exerted a significant therapeutic effect against RT-induced esophagitis (Koukourakis et al. 1999). Zhang et al. (2007) reported that oral administration of rhGM-CSF expressed in silkworm pupae (BmrhGM-CSF) had similar effects to that of an injection (Zhang et al. 2006). Since oral treatment with purified rhGM-CSF is effective, it would be interesting to know whether oral administration of unprocessed rhGM-CSF expressed in edible tissue could achieve the same effect. If this was proven, the cost could be drastically reduced. At present, the primary source of rhGM-CSF used in clinical applications is from recombinant DNA technology, including COS (Wong et al. 1985) and Namalwa cells (Okamoto et al. 1990) as well as yeast (Cantrell et al. 1985). However, these animal and microbial expression systems are costly to maintain, and more importantly, these systems are prone to potential viral contamination. Since the first successful case of the expression of a monoclonal antibody in plants was achieved (Hiatt et al. 1989), higher plants have provided us with a safe and economical way of producing plant-made pharmaceuticals (PMP). Many attempts have been made to express rhGM-CSF in plants, however, these systems have relatively low expression levels (Sardana et al. 2002; Shin et al. 2003; Wang et al. 2005). In those systems, the storage of recombinant proteins in the cytosol was limited by the activity of protease. However, cereal crop endosperm offers a unique way to store proteins in large quantities in specialized cell compartments. There is increasing evidence to indicate that cereal crop endosperm is one of the best systems to express and store large amounts of recombinant proteins (Yang et al. 2003; Yang et al. 2001). In the work described here, we present a rhGMCSF expression system in rice endosperm which achieved a high recombinant expression level and was effective in treating leucopenia by the oral administration of unprocessed transgenic grains. High expression levels of rice seed-based rhGM-CSF (OsrhGM-CSF) in rice endosperm was obtained by using a stronger endosperm-specific promoter and genetic codon optimization to maximize the transcription and translation efficiency, and a signal peptide was used to target OsrhGM-CSF to the protein bodies where OsrhGM-CSF could escape

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Biotechnol Lett (2008) 30:1679–1686

attack by protease in the cytosol. Oral administration of transgenic seeds to treat leucopenic mice resulted in an effective increase in the number of leukocytes in non-leucopenic mice and in leucopenic mice. This illustrates a novel approach in the use of oral unprocessed transgenic grains.

Materials and methods Plasmid construction All enzymes used in the study were purchased from New England Biolabs. Gt13a promoter with its signal peptide (Genebank accession No. AP003256) was amplified from genomic DNA of Taipei 309 (Oryza sativa subsp. japonica) using a forward primer, 50 -cccaagcttCAT GAGTAATGTGTGA-GCATTATGGGA-30 and a reverse primer 50 -cggtccgggcATTTGAATGTTAA GATAGAGTTTGT-30 . A 1284bp fragment was cloned into pUC18, and the resulting plasmid was designated pOsPMP02 (Gt13a-sp-Stuff-Nos). A human granulocyte-macro-phage colony stimulating factor (hgm-csf) gene (GeneBank accession No. CAA02621) was synthesized by Heron Blue Biotechnology Inc (Bothell, WA, USA) using rice-preferred genetic codons. The synthesized hgm-csf gene was then digested by SchI and XhoI, cloned into pOsPMP02 and the resulting construct was designated pOsPMP07 (Fig. 1a). Plasmid OsPMP05 containing a hygromycin phosphortransferase (hpt) gene driven by a callus-specific promoter from a rice b-cysteine protease (CP) gene (GeneBank accession No. AL732346) was used as a selective marker for rice transformation (Fig. 1a). Another two plasmids containing the beta-glucuronidase (gus) gene driven by Gt13a and Gt1 promoters, respectively were constructed for the GUS transient activity assay. The resulting plasmids were named pOsPMP41 (Gt13a-gusnos) and pOsPMP42 (Gt1-gus-nos) (Fig. 1a). For the determination of the tissue-specific expression of the Gt13a promoter, an expression cassette named pOsPMP45 containing Gt13a promoter/signal peptide, gfp gene and nos terminator was constructed (Fig. 1a). Finally, an entire expression cassette of 2218 bp fragment was digested by HindIII and EcoRI from pOsPMP45 and was inserted into the multiple cloning site of pCAMBIA1301. The final binary vector was transferred into Agrobacterium tumefaciens strain EHA105 by electroporation.


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Fig. 1 Various expression vectors used in this study (a), the assay of transcriptional activity (b) and tissue-specific analysis of Gt13a promoter (c). (a) pOsPMP07 consists of a Gt13a promoter and its signal peptide fused to a hgm-csf gene followed by a nos terminator; pOsPMP05 consists of a cysteine protease (CP) promoter, a hygromycin phosphotransferase (hpt) gene and nos terminator; pOsPMP41 and pOsPMP42 consist of a Gt13a and Gt1 promoter fused to gus gene and nos terminator; pOsPMP45 contained Gt13a promoter/signal

peptide, a gfp gene with a nos terminator. (b) The transcription activity of the two promoters was assayed by GUS transient analysis, the absolute value of luciferase activity of Gt1 and Gt13a were 1098 ± 24 and 870 ± 34, respectively; the absolute value of GUS activity was 0.073 ± 0.0006 and 0.063 ± 0.0006, respectively. (c) Tissue-specific analysis of Gt13a promoter by GFP transgenic plant. The green fluorescence can be detected in the endosperm only but not in leaf (L), root (R), stem (St), or embryo

Transient expression assay

using the callus regenerated from a rice variety TP309 as described previously (Yang et al. 2001). GFP transgenic plants were obtained through Agrobacterium transformation. Transgenic plants were transplanted and grown in a greenhouse to maturation.

Immature rice endosperms from 10–14 days after pollination (DAP) of 9311 (O. sativa subsp. indica) and plasmids of pOsPMP41, pOsPMP42 and pAHC18 as an internal control were used for the transient assay which was carried out according to the procedure described elsewhere (Yang et al. 2001). GUS activity was measured by reading at OD540 and the luciferase activity was measured by reading the relative light unit using SpectraMax M2 (Molecular Device). GUS activity was normalized to the luciferase activity. Genetic transformation Co-transformation of pOsPMP05 and pOsPMP07 was performed by micro-projectile-mediated transformation

Western blot analysis and ELISA analysis Immature endosperms, 20 lg, were homogenized using 500 ll protein extraction buffer (66 mM Tris/ HCl, pH 6.8, 2% SDS, 1 mM DTT) followed by centrifugation at 10,000 for 20 min. Protein concentration of the supernatant was determined using the DC Protein Assay Kit II. Proteins (10 lg) were loaded and separated by 15% (v/v) PAGE, and then transferred to nitrocellulose membranes according to the manufacturer’s instructions. A polyclonal rabbit

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antibody against hGM-CSF (US Biological) with 1:2,500 dilution and a goat anti-rabbit IgG conjugated with horseradish peroxidase (KPL) with a dilution of 1:7,500 were used for Western blotting. Finally, the blots were visualized by the 3, 30 -diaminobenzidine system. For ELISA analysis, total soluble protein (TSP) was extracted from 20 mg transgenic brown rice by homogenization in 500 ll of extraction buffer (50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 0.2% Tween 20). The extract was centrifuged (14,000 g) at 4°C for 30 min and the supernatant was used for quantification of rhGM-CSF using a human GM-CSF ELISA kit (U-cytech Bioscience) following the manufacturer’s instructions.


Kunming mice (25 ± 2 g) from the Animal Experimental Center of Wuhan University were assigned to four groups (n = 10), two groups were subcutaneously injected with 200 mg cyclophosphamide (CY)/ kg for three days, and then fed the transgenic rice seeds or the NT seeds for two weeks; the other two groups were subcutaneously injected with physiological saline (PS) for three days and then fed with the transgenic or NT seeds for two weeks. Leukocyte numbers were measured before CY or PS treatments and after 14 days feeding using a Syemex-KX21 trimodal globulimeter. All procedures were performed under an approved protocol in compliance with guidelines set by the Animal Experimental Center of Wuhan University.

Immuno-transmission electron microscopy Results Immature endosperms were harvested at 10–12 days after planting (DAP). Fixation, section preparation and immuno-electron microscopic observations followed the procedures described previously (Yang et al. 2003). The primary antibody (1:20 dilution) was the same as that used for the Western blot analysis and the secondary antibody (1:60 dilution) was a goat antirabbit IgG conjugated with gold (Sigma). GM-CSF biological activity assay TF-1 cells were provided by China Center for Type Culture Collection, and the protein was extracted using the same buffer as used in the ELISA analysis. All procedures followed those previously described (Sardana et al. 2002). Finally, 100 ll RPMI medium with 10% (v/v) fetal bovine serum (FBS) containing one of the following samples was added to each of the wells: 1 ng/ml commercial rhGM-CSF (E. coliderived), the transgenic rice seed extract containing 1 ng/ml of OsrhGM-CSF, the extract from the nontransgenic (NT) seeds at equivalent protein concentration, 1 ng/ml commercial hGM-CSF spiked with the NT seed extract, and the protein extraction buffer only. Live cells were counted by SpectraMax M2 using MTT after 96 h incubation. Mouse feeding experiment Mice fed with either 50% transgenic rice seeds or NT seeds were prepared for this study. Forty male

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Cloning of an endosperm-specific promoter Glutelin, a major seed storage protein, accounts for about 60% of the total protein in rice endosperm. Glutelin is encoded by a multigene family that consists of the GluA and GluB subfamilies. A study by Kusaba et al. (2003) showed that during twodimensional electrophoresis of rice seed proteins the glu1Ap3a (Gt13a) spot was the most remarkable which indicated that the promoter of Gt13a would have much higher transcription activity than that of other GluA subfamily members. To confirm this possibility we compared the activity of Gt13a and Gt1 promoters using the GUS transient assay, the Gt1 promoter was used to guide recombinant protein expression in previous studies. As shown in Fig. 1b, the GUS activity driven by the Gt13a promoter was 45% higher than that of the Gt1 promoter, consistent with the results of Kusaba et al. (2003). To confirm endosperm-specific expression of Gt13a promoter, G13a-sp-gfp cassette was transformed into Zhonghua11 (O. sativa subsp. japonica) genome and transgenic plants expressing GFP were obtained. The results showed that the green fluorescence could be detected in the endosperm, but not in the root, leaf, stem or embryo (Fig. 1c), indicating that Gt13a promoter is stringently endosperm-specific. Taken together, these results suggest that the Gt13a promoter possesses higher transcription activity and endosperm-specific expression.


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Generation of rice transgenic lines expressing hGM-CSF

Subcellular localization of rhGM-CSF in rice endosperm cells

Two plasmids, OsPMP07 and OsPMP05 were co-transformed into rice genome via micro-projectile-mediated transformation. In total, 12 independent transgenic plants were generated. These transgenic plants expressing OsrhGM-CSF were then screened by Western blot analysis. A distinct band of approximately 18 kDa with the same mobility as E. coliderived rhGM-CSF was observed in 6 lines but not in the NT seed extract. In addition, other bands ranging from 19–100 kDa in size were also observed in the transgenic lines (Fig. 2a). This suggested that OsrhGM-CSF could somehow be modified in rice endosperm cells. To determine the expression level, OsrhGM-CSF quantification was performed by ELISA. The expression level of OsrhGM-CSF ranged from 0.5 lg/seed (line 7–5) to 14 lg/seed (line 7–24) (Fig. 2b). Line 7–24, with the highest expression level was advanced to the next generation for further study.

In rice endosperm cells, there are two types of storage protein bodies, namely protein body I (PB I) and protein body II (PB II), which originated from the Endoplasmic Reticulum(ER) and the protein storage vacuole (PSV), respectively. To determine the subcellular localization of OsrhGM-CSF in transgenic endosperm cells, immunogold microscopic analysis was performed. As shown in Fig. 3, the majority of OsrhGM-CSF was found in PB I (Fig. 3a), in PB II (Fig. 3b), some OsrhGM-CSF was found in the apoplast (Fig. 3c) but not in the ER lumen (Fig. 3d). To rule out the possibility that the labeling in the apoplast was non-specific, higher dilution was tried (primary antibody 1:50 diluted, secondary antibody 1:150 diluted). Similar localization of OsrhGM-CSF was found with higher dilution, only the labeled particles were fewer (data not shown). In addition, a remarkable morphological change in both PB I and the ER was observed in the transgenic endosperm cells (Fig. 3e). Compared with NT endosperm cells (Fig. 3f), more PB I of smaller size was observed in the transgenic endosperm cells. Some PB I was retained in the ER lumen, which was rarely found in the NT endosperm cells. The morphology of PB II was not affected. Biological activity assay of OsrhGM-CSF

Fig. 2 Western blot and ELISA analyses of rhGM-CSF from T1 seeds of the transgenic lines. (a) Western analysis of T1 seeds from six independent transgenic plants. Lane 1: molecular mass; lane 2: rhGM-CSF derived from E. coli; lanes 3–8 are transgenic lines 7–5, 7–7, 7–8, 7–10, 7–12 and 7–24, respectively; lane 9 is the negative control of nontransgenic seeds of TP309; (b) quantification analysis of OsrhGM-CSF in those transgenic lines by ELISA

To further determine whether OsrhGM-CSF expressed in rice endosperm was biologically active, a cell culture study was carried out using TF-1 cells, a factor-dependent human erythroleukemic cell line which grows only in medium supplemented with GM-CSF or other growth factors. TF-1 cells were dispensed in eight replicates into a 96-well cell culture plates as described in Materials and methods. After 96 h incubation with different supplements, the number of live cells was measured. The results showed that rice seed-based OsrhGM-CSF had the same stimulatory effect on TF-1 cell proliferation as rhGM-CSF derived from E. coli (Fig. 4). Additionally, we found that the rice seed extract significantly inhibited the growth of TF-1 cells by 49.32% (t = 0.0016) when E. coli-derived rhGM-CSF was spiked with non-transgenic crude extracts. This suggested that unknown inhibitory substances may

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Fig. 3 Subcellular localization of OsrhGM-CSF in transgenic endosperm cells. (a) OsrhGM-CSF is readily seen in PB I in the transgenic endosperm cells; (b) OsrhGM-CSF can also be found in PB II; (c) OsrhGM-CSF is distributed throughout the apoplast; (d) the PB I-like structures remain in abnormal ER lumen, where the gold particles are localized in PB I-like structures but not in the ER lumen (arrow); panel e and f represent a lower magnification of the morphologic change of


PBs in the transgenic endosperm cells (e) but not in nontransgenic endosperm cells (f); some PB I-like structures remain in the ER lumen (arrowhead), which is rarely found in non-transgenic endosperm cells. PB I = protein body I, PB II = protein body II, SG = starch granule, ERM = endoplasmic reticulum membrane; magnification: (a) 33,0009; (b) 33,0009; (c) 43,0009; (d) 33,000; (e) 7,5009; (f) 7,5009

The effects of OsrhGM-CSF on mouse leukocytes

Fig. 4 OsrhGM-CSF can stimulate the proliferation of TF-1 cells. Cells were incubated in the presence or absence of seed extracts from transgenic and non-transgenic plants, extraction buffer only, and with E. coli-derived rhGM-CSF. Cell proliferation was measured by MTT

exist in rice seed extract which somehow inhibit TF-1 cell proliferation. Taken together, we conclude that OsrhGM-CSF produced in rice endosperm is biologically active and can effectively stimulate the proliferation of TF-1 cells.

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Cyclophosphamide (CY) is a drug used in the treatment of various cancers. Normal and cancerous cells are simultaneously affected by CY, resulting in serious side effects including a marked reduction in the production of leukocytes leading to decreased patient immunity. In order to alleviate these symptoms, rhGMCSF is administered in combination with CY treatment (Buisman et al. 1998; Gamba-Vitalo et al. 1991). To determine whether OsrhGM-CSF in unprocessed rice seeds can increase leukocyte numbers, an experiment using mice fed with rice seeds was performed. To create a leucopenia model, two groups of mice were subcutaneously injected with CY for three days, the number of leucocytes was observed to decrease dramatically (only 1.2 9 109/l) at 24 h post the last CY administration, which indicated that the leucopenia model was successfully established. In the leucopenic mice fed transgenic rice seeds for 14 days, the number of leukocytes significantly increased from 1.2 9 109/l to 9.4 9 109/l, while the number of leukocytes only


Fig. 5 OsrhGM-CSF can increase leukocyte numbers in nonleucopenic and leucopenic mice by directly feeding the animals with transgenic seeds. Mice were treated with cyclophosphamide (CY) or physiological saline (PS) for 3 days, respectively. Then mice were fed either transgenic seeds or non-transgenic (NT) seeds. Blood samples were collected for routine detection at the indicated time points

returned to a normal level (6.8 9 109/l) in mice fed NT seeds (Fig. 5). Mice fed transgenic seeds showed a 27% (t = 0.021) increase in leukocytes. In nonleucopenic mice, there was no significant change in the number of leukocytes after feeding with NT seeds, while the number of leukocytes increased to 10.5 9 109/l in mice fed transgenic seeds. This increase in leukocytes was 37% (t = 0.002) more than that in the NT seed feeding group. These results demonstrate that oral administration of unprocessed rice seed-based OsrhGM-CSF can effectively promote the proliferation of leukocytes in both non-leucopenic and leucopenic mice.

Discussion Up to now, extraordinary efforts have been made to improve rhGM-CSF yield in various plant species and some progress has been made. However, the expression level is still not high enough to be economically viable. We have successfully obtained a very high expression level (14 lg/seed) of OsrhGM-CSF in rice endosperm, which is the highest expression level reported in any plant system (Sardana et al. 2007). To improve the expression level of OsrhGM-CSF in rice endosperm cells, we chose a stronger, endosperm-specific promoter and rice-preferred codons to improve the transcription and translation efficiency, respectively. In addition, we used the signal peptide to guide OsrhGM-CSF into the endomembrane system of rice endosperm,

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such as the ER and the protein bodies, where OsrhGM-CSF could escape the attack of proteases in the cytosol and accumulate. There is increasing evidence to indicate that cereal crop endosperm is one of best expression systems for molecular pharming (Yang et al. 2001, 2003). Rice endosperm cells play a role in nutrient storage for seed germination, thus providing a favorite location for recombinant protein accumulation where they can escape from protease attack in the cytosol during seed development. Although the OsrhGM-CSF in our study was not only found in protein bodies but was also distributed throughout the apoplast in endosperm cells, the high expression level demonstrated that rice endosperm cells are safe for recombinant proteins. This may be because rice endosperm cells will enter into a self-destructive stage (apoptosis) after starch and proteins have accumulated. High-level expression of recombinant proteins (and their accumulation in the protein bodies) would not impose any physiological pressure on these cells. On the other hand, recombinant proteins in grain can be stored for 2–3 years in ideal temperature and moisture conditions, which would allow extensive transport and storage options which are not available in other plant organs. Oral delivery of pharmaceuticals is considered to be an attractive alternative to injections due to low cost and ease of use. Rice seeds, therefore, offer significant new opportunities for the production of safe and effective oral pharmaceuticals or vaccines. Human lysozyme and lactoferrin, for example, expressed in rice seeds have been successfully used as an oral supplement of rehydration solution to effectively reduce the duration of diarrhea by 30% in children (Zavaleta et al. 2007). Previous studies have shown that BmrhGM-CSF expressed in silkworm pupae with a molecular weight of 29 kDa was absorbed in the gut within 15 min of oral administration, and showed a double-peaked metabolic curve (Zhang et al. 2006). BmrhGM-CSF exerts biological activity in a dose-dependent manner, which is identical to that of rhGM-CSF by injection. Immunohistochemistry has demonstrated that BmrhGMCSF can pass through the small intestine mucosal wall into the bloodstream of mice, implying that rhGM-CSF can be absorbed as a functional molecule. In our study, OsrhGM-CSF expressed in rice seeds was much smaller than that in silkworm pupae. This may imply that the OsrhGM-CSF in our study could

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pass through the small intestine mucosal wall into the bloodstream of mice. Although the mechanism of leukocyte proliferation promotion via oral administration remains to be determined, the significance of unprocessed transgenic seed efficacy by oral administration cannot be overlooked. Considering the concerns of safety, efficacy, cost and the risk-benefit ratio of injection over oral administration, this study provides a novel, simple approach to treating various diseases by the oral administration of unprocessed transgenic grains. Acknowledgements We would like to thank Dr. Ning Huang for kindly providing the plasmid pAHC28 and Professor Jingping Ouyang for providing technical supports of the mice feeding experiment. This work is supported by the Project of the National ‘‘863’’ high technology Program in China (No. 2007AA100505), and the Key grant Project of the Chinese Ministry of Education (No. 307018).

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