A Comparison of Production Performance, Egg Quality, and Cecal Microbiota in Laying Hens Receiving Graded Levels of Vitamin B12

The objective of the study was to investigate the effect of fortified diets with standard vs. high levels of vitamin B12 on cecal microbiota composition, production performance, and eggshell quality of laying hens. Dietary treatments consisted of a basal diet with no supplementation of vitamin B12 or supplemented with 25, 100, and 400 μg/kg vitamin B12, respectively. A total of 432 laying hens were randomly assigned to four treatments with six replicates per treatment. No significant effect of dietary treatments on the production performance of hens was detected. The shell thickness of eggs from hens fed diet supplemented with 100 μg/kg of vitamin B12 was higher (P < 0.01) than that of eggs from hens fed control diet or supplemented with 25 μg/kg vitamin B12. The shell percentage of eggs from hens fed diet supplemented with 400 μg/kg of vitamin B12 was higher (P < 0.01) than that of eggs from hens fed other treatment diets. Dietary vitamin B12 did not modulate diversity of the cecal microbiota of the layers. At genus level, the cecal content from layers fed diet with supplemental level of 100 or 400 μg/kg of vitamin B12 had higher (P < 0.01) abundance of Faecalibacterium and lower (P < 0.05) abundance of Acinetobacter compared with the cecal content from layers fed other two diets. The abundance of Lactobacillus in the cecal samples from layers fed 100 μg/kg of supplemental level of vitamin B12 was higher (P < 0.05) than that from layers fed other three diets. The abundance of Butyricicoccus was higher (P < 0.05), while Bilophila was lower (P < 0.05) in the cecal content of layers fed 400 μg/kg of vitamin B12 diet compared with those from layers fed other three diets. The results of PICRUSt analysis indicated that 10 predicted metabolic functions of the cecal microbial communities were positively correlated to dietary vitamin B12 level. Overall, dietary supplementation of 100 or 400 μg/kg of vitamin B12 had equivalent effects and caused the significant change in composition and metabolic functions of cecal microorganisms, which could positively impact eggshell quality, metabolism, and gut health of laying hens.

The objective of the study was to investigate the effect of fortified diets with standard vs. high levels of vitamin B 12 on cecal microbiota composition, production performance, and eggshell quality of laying hens. Dietary treatments consisted of a basal diet with no supplementation of vitamin B 12 or supplemented with 25, 100, and 400 µg/kg vitamin B 12 , respectively. A total of 432 laying hens were randomly assigned to four treatments with six replicates per treatment. No significant effect of dietary treatments on the production performance of hens was detected. The shell thickness of eggs from hens fed diet supplemented with 100 µg/kg of vitamin B 12 was higher (P < 0.01) than that of eggs from hens fed control diet or supplemented with 25 µg/kg vitamin B 12 . The shell percentage of eggs from hens fed diet supplemented with 400 µg/kg of vitamin B 12 was higher (P < 0.01) than that of eggs from hens fed other treatment diets. Dietary vitamin B 12 did not modulate diversity of the cecal microbiota of the layers. At genus level, the cecal content from layers fed diet with supplemental level of 100 or 400 µg/kg of vitamin B 12 had higher (P < 0.01) abundance of Faecalibacterium and lower (P < 0.05) abundance of Acinetobacter compared with the cecal content from layers fed other two diets. The abundance of Lactobacillus in the cecal samples from layers fed 100 µg/kg of supplemental level of vitamin B 12 was higher (P < 0.05) than that from layers fed other three diets. The abundance of Butyricicoccus was higher (P < 0.05), while Bilophila was lower (P < 0.05) in the cecal content of layers fed 400 µg/kg of vitamin B 12 diet compared with those from layers fed other three diets. The results of PICRUSt analysis indicated that 10 predicted metabolic functions of the cecal microbial communities were positively correlated to dietary vitamin B 12 level. Overall, dietary supplementation of 100 or 400 µg/kg of vitamin B 12 had equivalent effects and caused the significant change in composition and metabolic functions of cecal microorganisms, which could positively impact eggshell quality, metabolism, and gut health of laying hens.

INTRODUCTION
Vitamin B 12 is one of essential B vitamins required by humans and animals. Since it is involved in nucleic acid synthesis, carbohydrate and fat metabolism, and methyl synthesis, the deficiency of vitamin B 12 will cause anemia, muscle weakness, severe neurologic problems, and many other symptoms in humans and animals (1)(2)(3)(4)(5)(6). In laying hens, numerous studies indicated that vitamin B 12 is required for optimal egg production, egg weight, and hen weight (7)(8)(9)(10)(11). Today, vitamins are commonly over fed in poultry feed to give safety margin for the deterioration of many vitamins during feed processing and storage (12)(13)(14). Also, vitamin-enriched eggs produced through supplementing extremely a high level of vitamins of interest to the layer diet for human consumption have been widely accepted (15)(16)(17)(18)(19)(20). Subsequently, a significant portion of vitamins that was not absorbed in the small intestine of layers could reach the hind gut and affect the microbiota composition of the cecum. Giannella et al. reported that excess vitamin B 12 in the intestine changed the gut microbiome composition and caused the overgrowth of intestinal bacterial (21). Degnan et al. found that the competition and exchange of vitamin B 12 among microbes can regulate the gut microcommunity (22). It was documented that vitamin B 12 and some other B vitamins changed the metabolism of microbiota, promoted bacterial colonization in the gut, and modulated bacterial virulence and the host defense to the pathogen infection (23)(24)(25). Results from in vitro and in vivo trials indicated that vitamin B 12 was essential for some enteropathogens to utilize ethanolamine, which enhanced Salmonella typhimurium growth and its virulence gene expression (26)(27)(28)(29). Xu et al. indicated that vitamin B 12 supplementation changed microbial composition and increased the amount of short-chain fatty acids in an in vitro colon simulation (30).
Thus far, not much information is available for the link between high dietary vitamin B 12 and the intestinal microbiota composition in poultry. Therefore, the purpose of the current study was to investigate the effect of fortified diets with standard vs. high levels of vitamin B 12 on cecal microbiota composition and production performance of laying hens.

METHODS AND MATERIALS
All experimental procedures were carried out in accordance with the Guidelines of the Shanxi Agricultural University Animal Experiment Ethics Committee, and the license number was SXAU-EAW-2017-002Chi.001. The experiment was performed in the Animal Production Laboratory of Shanxi Agricultural University and Xingmin Animal Husbandry Industry Cooperative located in Taigu District of Jinzhong City.

Animals, Diets, and Experimental Design
Based on a single-factor experimental design, a total of 432 healthy Jinghong No. 1 laying hens with the same initial body weight (BW) at 30 weeks of age were randomly assigned to four dietary treatments to give six replicate cages of three hens per cage. Dietary treatments included (1) corn soybean  12 was detected in the basal diet by using high-performance liquid chromatography. This is because vitamin B 12 is well-known to be the sole vitamin that is absent from plant-based feed sources. Three dietary supplemental levels of vitamin B 12 included one standard level of 25 µg/kg recommended by breeders and vitamin-producing companies (31,32), and two (100 or 400 µg/kg) high levels were used for producing vitamin B 12 -enriched eggs (15,16). Other nutrient level of basal diet was designed based on the NRC recommendation (33). The composition and calculated nutrient level of basal diet are listed in Table 1.
The experimental chicken coop was completely enclosed with the stainless-steel galvanized cages of 47 * 37 * 38 cm in size. In a 14-day pretrial period, all laying hens were fed a basal diet. Then the treatment diets were provided to the laying hens for another 42 days. The layers were free to eat feed and drink water during the entire pretrial and trial periods. The daily light exposure was 16 h with an intensity not <15 lx/m 2 .
The Assay of Production Performance, Egg Quality, and Serum Indicators of Laying Hens Hens in each replicate were weighed in-group at the start and at the end of the experiment to evaluate the BW change. Egg number, egg weight, and dead birds were recorded daily, whereas feed consumption was recorded weekly to calculate hen day egg production (%), feed intake (g/hen/day), egg mass (g/day), and feed conversion ratio (feed/egg mass).
A total of 18 egg samples (three eggs per replicate) were taken weekly in a 6-week trial period to do the analysis of egg weight and eggshell quality. Egg diameter was measured with a digital display vernier caliper (S102-101-101, SMCT, Shanghai). Eggshell strength was evaluated using an Egg Force Reader (EFR-01, Orka Food Technology Ltd., Israel). Eggshell thickness was measured on the large end, equatorial region, and small end, respectively, using an eggshell thickness gauge (Robotmation Co., Ltd., Tokyo, Japan), and the average value was calculated as the eggshell thickness measurement. The egg weight, egg yolk color, and Haugh unit (HU) were evaluated using an Egg Analyzer (EA-01, Orka Food Technology Ltd., Israel). Egg white and yolk were separated carefully, then the egg yolk and eggshell were weighed, and the ratio of the egg yolk, egg white, and eggshell was calculated.
Blood samples were taken from six hens per treatment (one bird per cage) to test the biochemical indicators of serum. Vitamin B 12 , progesterone, and estrogen levels were measured using enzyme-linked immunoassay kit (Shanghai Hushen Biological Technology Co., Ltd., China). Total cholesterol (TC) and triglyceride (TG) levels were measured using GPO-PAP (NanJing JianCheng Bioengineering Institute, China).

The Assay of Cecal Microbiome
At the end of the experiment, one bird with a BW close to the mean BW of each cage was euthanized by cervical dislocation to collect the cecal content. A total of six samples per treatment were collected. After collection, the samples were immediately frozen by liquid nitrogen and preserved at −80 • C until analysis. The frozen samples were used for isolation of metagenomic DNA.
Thermal cycling included initial denaturation at 98 • C for 2 min, followed by 25 cycles consisting of denaturation at 98 • C for 15 s, annealing at 55 • C for 30 s, and extension at 72 • C for 30 s, with a final extension of 5 min at 72 • C. PCR amplicons were purified with Agencourt AMPure Beads (Beckman Coulter, Indianapolis, IN, USA) and quantified using the PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA). After the individual quantification step, amplicons were pooled in equal amounts, and paired-end 2,300-bp sequencing was performed using the Illlumina MiSeq platform with MiSeq Reagent Kit v3 at Shanghai Personal Biotechnology Co., Ltd. (Shanghai, China).

Sequence Analysis
The Quantitative Insights Into Microbial Ecology (QIIME, v1.8.0) pipeline was employed to process the sequencing data, as previously described (34). Briefly, raw sequencing reads with exact matches to the barcodes were assigned to respective samples and identified as valid sequences. The low-quality sequences were filtered through following criteria (35,36) sequences that had a length of <150 bp, sequences that had average Phred scores of <20, sequences that contained ambiguous bases, and sequences that contained mononucleotide repeats of >8 bp. Paired-end reads were assembled using FLASH (37). After chimera detection, the remaining high-quality sequences were clustered into operational taxonomic units (OTUs) at 97% sequence identity by UCLUST (38). A representative sequence was selected from each OTU using default parameters. OTU taxonomic classification was conducted by BLAST searching the representative sequences set against the Greengenes Database (39) using the best hit (40).

Bioinformatics and Statistical Analysis
Sequence data analyses were mainly performed using QIIME and R packages (v3.2.0). OTU-level alpha diversity indices, such as Chao1 richness estimator, ACE metric (Abundancebased Coverage Estimator), Shannon diversity index, and Simpson index, were calculated using the OTU table in QIIME. OTU-level ranked abundance curves were generated to compare the richness and evenness of OTUs among samples. Differences in the Unifrac distances for pairwise comparisons among groups were determined using Student's t-test and the Monte Carlo permutation test with 1,000 permutations. The taxonomy compositions and abundances were visualized using MEGAN (41) and GraPhlAn (42). Venn diagram was generated to visualize the shared and unique OTUs among samples or groups using R package "VennDiagram, " based on the occurrence of OTUs across samples/groups regardless of their relative abundance (43). Taxa abundances at the phylum and genus levels were statistically compared among samples or groups by Metastats (44). Pattern Search was used to identify correlation between the microbial composition of cecal content and dietary supplemental level of vitamin B 12 . Microbial functions were predicted by PICRUSt (phylogenetic investigation of communities by reconstruction of unobserved states), based on high-quality sequences (45). Correlation Heatmap was performed using the OmicStudio tools at https:// www.omicstudio.cn/tool.
Data were analyzed with one-way ANOVA by using Statistical Product and Service Solutions (SPSS) 22.0 (SPSS Inc., Chicago, IL, USA). The significant difference of means among treatment

Production Performance, Egg Quality, and Serum Indicators of Laying Hens
Effects of dietary vitamin B 12 on the production performance of laying hens are shown in Table 2. There was no significant difference among all treatments for egg production, feed intake, feed conversion ratio, egg mass, and egg weight (P > 0.05). Table 3 lists the effect of dietary vitamin B 12 on egg quality parameters. The shell percentage of eggs from hens fed diet supplemented with 400 µg/kg of vitamin B 12 was higher (P < 0.05) than that of eggs from hens fed other treatment diets. The shell thickness of eggs from hens fed diet supplemented with 100 µg/kg of vitamin B 12 was higher (P < 0.05) than that of eggs from hens fed control diet or supplemented with 25 µg/kg of vitamin B 12 . No significant effect of dietary treatments on other egg quality parameters was detected (P > 0.05).
The biochemical indicators of serum are listed in Table 4. The vitamin B 12 concentration of serum from hens fed diet with a Frontiers in Veterinary Science | www.frontiersin.org supplementation of 25 µg/kg of vitamin B 12 was higher (P < 0.05) than that from hens fed diet with no supplementation of vitamin B 12 , but lower (P < 0.05) than that from hens fed other two diets with high supplemental levels of vitamin B 12 . There was no significant difference among all treatments for other parameters.

Cecal Microbiota Composition
To explore the diversity of the cecal microbiota of layers after dietary vitamin B 12 supplementation, the composition and species distribution of the cecal microbiota were investigated by 16S rRNA gene sequencing. After removal of the questioning sequences, a total sequencing quantity was 952,855 that was from 24 samples of cecal content with an average of 39,702 sequences per sample (range from 27,769 to 50,808) for subsequent analysis. A total of 4,386 OTUs after streamlining was characterized into different taxonomic levels including phylum, class, order, family, genus, and species based on Green gene database through QIIME with a 97% species similarity. Venn diagram analysis showed that 1,927 OTUs were shared among the four dietary treatment groups (Figure 1). There were 3,413, 3,165, 3,446, and 3,211 OTUs in treatments B0, B25, B100, and B400, respectively. These results indicated that dietary vitamin B 12 did not modulate diversity of the cecal microbiota of the layers. However, there were 141, 92, 104, and 83 OUTs that were uniquely identified in four different treatments.
The rarefaction curves and species accumulation curve (Figures 2A,B) for each sample leveled off as the number of sequences increased in all four treatment groups indicating that the samples analyzed had sufficient sequence coverage to accurately describe the bacterial composition of the cecal content in this study.
The alpha diversity of microbiota in the cecal content, including species diversity (Shannon and Simpson indices) and richness (ACE and Chao 1 indices), is shown in Table 5. There was no significant difference among all treatments for these indices, although the group with no vitamin B 12 supplementation tended to have higher values of ACE and Chao1. Figure 3A and Supplementary Table 1 demonstrated the microbial composition of cecal content from four dietary treatments in the phylum level. The results indicated that cecal microbiota of layers was mainly composed of Firmicutes, Bacteroidetes, and Proteobacteria accounting for about 94%, with Firmicutes being the predominant phylum (>52). A numerical shift in the proportion of Bacteroidetes and Proteobacteria to Firmicutes was observed when high-level vitamin B 12 (100 or 400 µg/kg) was included in the diets. The proportion of Firmicutes, Actinobacteria, Deferribacteres, WPS_2, and Spirochaetes is positively correlated, and the proportion of Elusimicrobia, Thermi, TM7, Cyanobacteria, Lentisphaerae, Planctomycetes, Proteobacteria, Fusobacteria, Verrucomicrobia, Synergistetes, and Bacteroidetes is negatively correlated with the supplemental level of vitamin B 12 (Figure 3C).
At genus level, a total of 173 species of bacteria in the cecal content of laying hens was observed in this trial, among which 17 genera had the relative abundance of more than 1% (Figure 3B; Supplementary Table 2). Pattern Search results   indicated that the abundance of Butyricicoccus, Faecalibacterium, Paludibacter, p_75_a5, and Unclassified_Ruminococcaceae has a positive correlation, and the abundance of the other 20 bacteria has a negative correlation with the dietary supplemental level of vitamin B 12 ( Figure 3D). As shown in Figure 4, the cecal content from layers fed a diet with supplemental level of 100 or 400 µg/kg of vitamin B 12 had higher (P < 0.01) abundance of Faecalibacterium and lower (P < 0.05) abundance of Acinetobacter compared with the cecal content from layers fed other two diets. The abundance of Lactobacillus in the cecal samples from layers fed 100 µg/kg of a supplemental level of vitamin B 12 was higher (P < 0.05) than that from layers fed with the other three diets. The abundance of Paraprevotella, Blvii28, Perlucidibaca, and Succinatimonas in the cecal content of the layers fed diets supplemented with 25 or 100 µg/kg of vitamin B 12 was higher (P < 0.05) than that from the layers fed with the other two diets. It was worth noting that Paludibacter was not detected in the cecal content of layers fed a diet with no supplementation of vitamin B 12 . The abundance of Butyricicoccus was significantly higher, while Bilophila was significantly lower in the cecal content of layers fed 400 µg/kg of vitamin B 12 diet compared with that from layers fed with the other three diets.

Functional Prediction of the Gut Microbiota
In order to get the predicted metabolic functions of the cecal microbial communities from different treatments, PICRUSt analysis was performed. The results are shown in Figure 5. Ten predicted metabolic functions of the cecal microbial communities were positively correlated with dietary vitamin B 12 level (P < 0.05). These metabolic functions included (1) carbohydrate metabolism such as starch and sucrose metabolism, pentose and glucuronate interconversions, other glycan degradation, and galactose metabolism; (2) lipid metabolism such as sphingolipid metabolism and secondary bile acid biosynthesis; (3) biosynthesis of other secondary metabolites such as phenylpropanoid biosynthesis and flavone and flavonol biosynthesis; (4) other metabolisms such as polycyclic aromatic hydrocarbon degradation and biosynthesis and biodegradation of secondary metabolites. The cecal microbiota from layers fed a diet with 400 µg/kg of vitamin B 12 increased all the predicted metabolic functions except flavone and flavonol biosynthesis.
Based on the results of Spearman's analysis (Figure 6), a positive correlation (P < 0.05) between the abundance of Acinetobacter in the cecal content and the biosynthesis and biodegradation of secondary metabolites and polycyclic aromatic hydrocarbon degradation was found. The abundance of Butyricicoccus and Paludibacter in the cecal content was positively correlated (P < 0.05) with carbohydrate and lipid metabolism. A negative correlation (P < 0.05) between the abundance of Bilophila in the cecal content and biosynthesis and biodegradation of secondary metabolites was observed. The abundance of Lactobacillus was positively correlated (P < 0.05) with flavone and flavonol biosynthesis.

DISCUSSION
The results from the current study showed no significant effect of dietary vitamin B 12 levels on the production performance of laying hens including egg production, feed intake, and egg weight. Although the concentration of vitamin B 12 in B0 is below the detectable limit, no negative impact on the production performance due to deficiency of vitamin B 12 was noticed. This is probably because the layers might get enough reservation of vitamin B 12 in the tissues from pretrial diets to support the need of production. The similar results were reported by other researchers (46)(47)(48). Kominato reported that 2-5 months may be needed to completely deplete hens of vitamin B 12 stored in tissues (49). The high dietary levels of vitamin B 12 (100 or 400 µg/kg) used in this trial did not affect the production performance of layers, but significantly increased the thickness and the percentage of eggshell. Leeson and Caston reported that a high dietary vitamin B 12 had no effect on the egg production of laying hens (15). The improvement of eggshell quality is probably due to the change in microbiota and their metabolites in the gut. A positive correlation between the abundance of butyrateproducing bacterium and Lactobacillus in the cecal content and dietary vitamin B 12 level was found in this trail. Many studies indicated the beneficial effect of dietary inclusion of butyrate or probiotic including Lactobacillus on the eggshell quality (50-53). Vitamin B 12 is a critical nutrient for human beings, animals, as well as microbes. Studies indicated that dietary inclusion of vitamin B 12 increased the level of vitamin B 12 in the hind gut and modulated the structure and function of microbial communities in humans and mice (22,54,55). In the current study, although different dietary levels of vitamin B 12 did not alter the diversity of cecal microbiota of layer hens as evident by the similar α-diversity index among different treatments, the community structure and abundance of the microbiota in the cecum were significantly changed. A positive correlation between dietary level of vitamin B 12 and cecal abundance of Firmicutes was detected. Studies found that Firmicutes is one of the dominant microorganisms in the cecum of chicken, and the abundance of Firmicutes is beneficial to the health of the bird because of its anti-inflammatory effects (56)(57)(58). The negative correlation between the dietary level of vitamin B 12 and the richness of Bacteroidetes in the cecal content observed in this trial did not support the results reported by Kelly et al. and Wang (55,59). The abundance changes in microbiota due to dietary vitamin B 12 level could impact the energy metabolism of laying hens. This is based on some evidence showing the close relationship between the abundance of Firmicutes or Bacteroidetes and body fat accumulation in humans and some obesity-related parameters in mice (60,61). However, further investigation is needed.
It is well-documented that butyrate can positively impact intestinal health through the synergistic effect of providing energy to the intestinal cell and anti-inflammatory effect (62)(63)(64). Major butyrate-producing bacteria in the gastral intestinal tract included F. prasusnitzii in Faecalibacterium genus and Butyricoccus (65,66). They all played a very important role in host intestinal health (58,(66)(67)(68). Results from the current study indicated that Faecalibacterium was the core genus in the cecum of laying hens and was positively correlated with dietary supplemental levels of vitamin B 12 , and dietary supplementation of high-level vitamin B 12 (100 or 400 µg/kg) increased the abundance of Butyricoccus. The increased abundance of Lactobacillus, Paraprevotella, Succinatimonas, Paludibacter, and Sphaerochaeta in the cecal content due to high dietary vitamin B 12 (100 or 400 µg/kg) observed in this trial could also positively impact the gut health of laying hens. This is because bacteria in the gut, including Lactobacillus and Paraprevotella, have been proven to play an active and protective role in intestinal health of the host through producing antimicrobial molecules, competitive exclusion, and changing bile acid metabolism in the gut (69)(70)(71). Succinatimonas, Paludibacter, and Sphaerochaeta were found to produce volatile fatty acids including acetic acid, propionic acid, and succinic acid that could positively impact energy metabolism of the host and regulate hindgut pH to inhibit the growth of harmful bacteria (72)(73)(74)(75)(76)(77). Dietary supplementation of high-level vitamin B 12 depressed the richness of Acinetobacter and Bilophila that were inflammation-associated genera (78). The composition changes in cecal microbiota of laying hens found in this trial due to high dietary vitamin B 12 was also significantly correlated to the predicted metabolism function of microbial communities in the cecum including carbohydrate, lipid, and FIGURE 6 | The correlation heatmap between the abundance of microbiota at genus level in the cecal content and the predicted metabolic functions. *P < 0.05, **P < 0.01. many other metabolisms. Similar results were reported by other researchers (79).

CONCLUSIONS
To our knowledge, this is the first trial to explore the relationship between high dietary levels of vitamin B 12 and the cecal microbiota in laying hens. The results indicated that dietary supplementation of 100 µg/kg of vitamin B 12 had equivalent effects with the supplemental level of 400 µg/kg. Both levels caused a significant change in composition and metabolic functions of cecal microorganisms, which could positively impact eggshell quality, metabolism, and gut health of laying hens during peak production period. These findings provided the valuable information for the application of high supplemental levels of vitamin B 12 in the diet of laying hens for production or health purposes.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: BioProject ID PRJNA732929.

ETHICS STATEMENT
All experimental procedures were carried out in accordance with the Guidelines of the Shanxi Agricultural University Animal Experiment Ethics Committee, and the license number was SXAU-EAW-2017-002Chi.001.

AUTHOR CONTRIBUTIONS
YY: conceptualized the study, supervised the study, was in charge of the project administration, and acquired the funding. RW: developed the methodology, performed the formal analysis and data curation, obtained the resources, and wrote and prepared the original draft. RRL and RW: conducted the investigation. RW, XTW, and YB wrote, reviewed, and edited the manuscript. All authors have read and agreed to the published version of the manuscript.

ACKNOWLEDGMENTS
We would like to thank relative personnel of Xingmin Animal Husbandry Cooperative (Taigu, China) for guidance in the feeding process. We would also like to thank Editage (www.editage.cn) for English language editing.