CRISPR/Cas9-based Knockout of miR-487b-3p Accelerates The Cell Proliferation of Primary Goat Myoblast by Relieving Its Inhibition of IRS1

Background: MicroRNAs (miRNAs) are endogenously expressed small noncoding RNAs with a length of about 22 nucleotides and play critical roles in the regulation of posttranscriptional gene expression. miR-487b-3p has been recently identied as a key regulator in skeletal muscle growth and development. However, the function of miR-487b-3p on goat skeletal muscle remains to be investigated. Results: In this study, we found that miR-487b-3p was widespread in goat different tissues, with a signicantly higher expression in muscle, especially in lamb. The results demonstrated that the expression of miR-487b-3p was gradually up-regulated during myoblast proliferation. Then, miR-487b-3p knockout primary goat myoblast cells clones were obtained by using CRISPR/Cas9 system and our RPG surrogate reporter-based screening. Further investigation uncovered that the knockout of miR-487b-3p signicantly accelerated primary goat myoblast proliferation, which was accompanied by up-regulation of cell cycle-related genes, such as PCNA, Cyclin E and CDK2. What’s more, we found that miR-487b-3p targets directly the 3'UTR of insulin receptor substrate 1 (IRS1) gene and IRS1 knockdown by siRNA was able to down-regulate the expression of the cell cycle-related genes. Conclusions: Collectively, these founding demonstrated miR-487b-3p as a potent inhibitor of cell proliferation which functions by targeting IRS1 gene in primary goat myoblast.


Background
Skeletal muscle is the most abundant tissue in mammals and plays an important role in body metabolism [1]. During the formation of skeletal muscle, mononucleated myoblast expand, migrate, and differentiate into myoblast, which is a complex process regulated by a group of myogenic regulator factors (MRF). These regulators, including Pax3, Pax7, Myf5 and MyoG, each of which plays an essential role in skeletal muscle development [2; 3]. Therefore, further understanding of the skeletal muscle development process and related molecular mechanisms in goat will contribute to improve the growth of young lambs and facilitate the breeding mutton goats.
MicroRNAs (miRNAs) are a class of evolutionarily conserved small RNAs that inhibit target gene expression at a post-transcriptional regulation either by degrading or arresting the translation of speci c messenger RNAs (mRNAs). The short single-stranded miRNA usually binds to the 3' untranslated region (3'UTR) of speci c mRNA based on sequence homology [4; 5], resulting in the degradation of target mRNA or the repression of corresponding protein translation [6]. Up to now, a series of miRNAs have been veri ed to be involved in the regulation of myoblast proliferation and differentiation. Among which, miR-1 [7], miR-133 [8], miR-206 and miR-486 [9] are key muscle speci c regulators involved in muscle myogenesis. In addition, skeletal muscle is also enriched with many ubiquitously expressed miRNAs, including miR-24 [10], miR-29 [11], miR-125b [12] miR-181 [13], and miR-214 [14], which are also necessary for the regulation of muscle development. Both muscle-speci c and ubiquitously expressed miRNAs have been demonstrated as major regulators of fundamental biological processes and play essential roles in myogenesis [15]. However, further study of functional miRNAs in goat muscle development, although which is just a small fraction of the many thousands of miRNAs, remains necessary to improve our understanding how miRNA net-work regulates goat myogenesis.
Previous studies have reported that the cluster 14q32.31 member miR-487b-3p is ubiquitously expressed and plays important roles in many biological processes, such as cell proliferation, differentiation, and disease [16; 17]. miR-487b binds directly to both rat and human insulin receptor substrate 1 (IRS1) 3'UTR and inhibits reporter gene expression in primary rat and human arterial adventitial broblasts [16; 18]. IRS1 is one of the dominant acting regulators of cell proliferation and survival. Recent study showed that miR-487b-3p expression varies from different stages of goat muscle development and myogenesis, and could delay proliferation and differentiation in mouse myoblast-derived C2C12 cells [16]. However, to our knowledge, the function of miR-487b-3p on goat myogenesis remains unknown.
Synthetic small interfering RNAs (siRNAs) are commonly used to knock-down target RNAs to explore their functions, including mRNAs, lncRNAs, miRNAs [19; 20] and so on. Despite it's fast and cost e cient, RNA interference (RNAi) technique suffers from limited short-term inhibition and inconsistent silencing e ciency. The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPRassociated protein 9) technology has been widely used for genome editing in various cell types and organisms since its advent [21]. With robust on-target activity and high delity, CRISPR/Cas9 technology can be used to knock-out interested miRNA by introducing indels into its seed sequence, and has become a more preferred tool than RNAi in the study of miRNA gene function [22]. Here, to investigate the role of miR-487b-3p in goat myogenesis, we created miR-487b-3p knockout (KO) primary goat myoblast cell clones by using CRISPR/Cas9 system and our DsRed-Puro r -eGFP (RPG) surrogate reporter-based screening [23][24][25]. Further experimental investigation demonstrated miR-487b-3p as a potent inhibitor of cells proliferation and that the knockout of miR-487b-3p signi cantly accelerated primary goat myoblast proliferation. In summary, this study provides new insight into the function of miR-487b-3p in primary goat myoblast, which will expand our understanding of its role in the regulation of skeletal muscle growth.

Goat tissue samples and primary myoblast cells
The goats were raised in strict accordance with the guides of Northwest Agricultural and Forestry University (NWAFU) Animal Care and Use Committee. Heart, liver, spleen, lung, kidney, and muscle tissue samples were collected from three lamb goat (90 days old) and three adult goats (2 years old). Primary myoblast cells were isolated from skeletal muscle tissues of the lamb goat, and were puri ed and cultured according to previously described protocol [26]. Brie y, cells were cultured in growth medium containing DMEM/F12 basic medium (C11330500BT, Invitrogen Corp, Waltham, MA, USA), 20% fetal bovine serum (FBS) (10099-141C, Invitrogen Corp., Waltham, MA, USA), and antibiotics (100 U/mL penicillin and 100 g/mL streptomycin) (15140-122, Invitrogen Corp., Waltham, MA, USA) in 5% CO 2 at 37℃, with the medium changed every day.

RNA Isolation and real-time quantitative PCR (RT-qPCR)
Total RNA was extracted from goat tissue samples or cultured cells using the reagent Trizol (9109, Takara). The concentration of total RNA measured by NanoDrop2000 (Thermo scienti c) and the quality was checked by denaturing agarose gel electrophoresis. The cDNA synthesis was performed with reverse transcription kits (RR047A, Perfect Real Time, Takara). For quanti cation of miR-487b-3p expression, miRNA speci c complementary DNA was generated using miRNA stem-loop-speci c primers ( Table 1). The cDNA generated was stored at -20°C for subsequent usage.
RT-qPCR was performed using TB green premix Ex taq II (RR820A, Perfect Real Time, Takara) on a Light Cycler 96 real-time system (Roche) with a 25 μL reaction volume. Each sample was carried out in triplicate and was repeated for three times at least. The mRNA expression levels of all coding genes were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which was used as an internal standard. On the other hand, the expression level of miR-487b was quanti ed with 18S RNA as the reference. The relative gene expression was analyzed using the comparative Ct (2 −△△Ct ) method.

Construction of CRISPR/Cas9 and RPG surrogate reporter vectors
In order to knockout miR-487b, three top single guide RNAs (sgRNAs) were designed according to the genomic sequence of Capra hircus miR-487b-3p (chi-miR-487b-3p) using the CRISPR Design Tool (http://chopchop.cbu.uib.no/) based on the following criteria: the sgRNA should have a high e ciency score and low off-target effects. Single-strand DNA annealing oligonucleotides for the guide and the target sequences of the three sgRNAs were synthesized by Invitrogen (Shanghai, China, as shown in table 2). Then, Annealed oligonucleotides for the three sgRNA guides were cloned into the Bbs I sites of the plasmid pX330-U6-Chimeric_BB-CBh-hSpCas9 (pX330) (plasmid #42230, Addgene, Cambridge, MA) to construct the corresponding sgRNA/Cas9 expression vectors. The SSA-RPG surrogate reporter vectors was designed and constructed as we previously reported [24]. Generally, the oligo-nucleotide annealed products with PAMs for the three sgRNA targets were inserted into the Not I/BamH I sites of the SSA-RPG parental vector to generate the corresponding surrogate reporter vectors, respectively. All the plasmid vectors were con rmed by sequencing.
Cell transfection, screening and T7E1 cleavage assays Primary goat myoblast cells (1×10 6 cells/well) were seeded into 6-well plates and cultured in normal culture medium to reach 80% of con uence. The myoblast cells were transiently transfected with 3 μg plasmid DNA (2 μg sgRNA/Cas9 expression vector and 1 μg RPG surrogate reporter vector) using Lipofectamine 3000 reagent (L3000008, Invitrogen, Waltham, MA) following to the manufacturer's protocol. The parental vector pX330 without sgRNA guide sequence served as a negative control. The medium was changed 4 hours after transfection and the cells were screened for another 48 hours with 1.0 μg/mL of puromycin (P8833, Sigma) supplemented. Surviving cells were expanded and the genomic DNAs were extracted using the E.Z.N.A.DNA Kit (OMEGA Bio-Tek) for PCR and T7E1 cleavage assay. PCR was conducted using the 2×Taq Master Mix (Novoprotein). The DNA fragments spanning target sites were ampli ed with the primers: F 5΄-GACCCAGTCCACATAC AGCAAG-3΄, R 5΄-CGATAATCGCATCACCATTAC-3΄. Puri ed PCR products were subject to T7E1-cleavage assay (M0302L, NEB, Ipswich, MA, USA), and the digested DNA fragments were detected by gel electrophoresis. The band intensity for different DNA fragments was measured by Image J software (Image Lab, http://imagej.net) and was used for calculating the indel frequencies within the target sites as reported previously [27]. The puri ed PCR products were also inserted into the pMD™19-T Vector (Takara) by TA cloning for sequencing veri cation.
In addition, for chemically synthesized siRNAs, cells were transfected at 50% density with 50 nM RNA using lipofectamine 3000. The siRNA sequences are shown in Supplementary table S1.

Myoblast cells clone detection and off-target effect analysis
After puromycin resistant screening, selected myoblast cells were seeded into a 96-well plate for single cell cloning. After 10-15 days further culture, the cells were collected with half of each cell clones were seeded into new 48-well plates and the remaining half for genomic DNA extraction. Then, the precursor sequence of miR-487b-3p were ampli ed by PCR for validating the genomic modi cation by sequencing.
To assess the off-target effects, the potential off-target sites (POTs) for each sgRNA were predicted according to an online design tool (http://crispr.mit.edu/) and Cas-OFFinder (http://www.rgenome.net/cas-o nder/). The genomic sequences of the POTs were also ampli ed by PCR for sequencing veri cation. The primer sequences are shown in Supplementary table S2.

Cell counting kit-8 (CCK-8) and EdU imaging assays
The miR-487b-3p KO myoblast cells were seeded at a density of 1×10 3 cells per well in a 96 well plate in growth medium. Six independent biological replicates for each treatment were conducted. After the maintained for 24 hours, the cells were switched to the medium with 10% Cell Counting Kit-8 (CCK-8) reagent (EQ829, DOJINDO) and were incubated for another 4 hours at 37℃, which was followed by the absorbance measurement at 450 nm using a SYNERGY/H1 microplate reader (BioTek). The intensity of the color was directly proportional to the number of viable cells in the sample.
In addition, EdU imaging assay was carried out using the 488 EdU Click Proliferation kit (Beyotime) according to the manufacturer's instruction. Myoblast cells were incubated with EdU (50 mM) (Beyotime) in the culture medium for 2 hours. Following incorporation of EdU and xation, myoblast cells were subjected to Click-iT reaction (Invitrogen) for 30 minutes in the dark to add biotin to the EdU, and the cell nuclei were stained with Hoechst 33342 for 10 minutes. Afterward, the cells were visualized by using a Cell imaging multifunctional detection system (BioTek) and the data were analyzed with Image J software.

Flow cytometric analysis
The miR-487b-3p KO myoblast cells were seeded in 6-well plates at a density of 5×10 5 cells per well. After 48-hour culture, cells were washed three times with PBS and harvested. The cells were collected and xed in cold 70% ethanol at 4 °C overnight. After RNase A treatment (1 mg/mL) at 37℃ for 30 minutes, and then staining with 4,6-diamidino-2-phenylindole (DAPI) staining solution (Solarbio) (50 µg/mL DAPI, 0.2% Triton X-100, 100 µg/mL RNase, PBS) for 1 hour at 4℃ in the dark. Then the cell suspension was centrifuged at 1200 rpm for 5 minutes, and the supernatant was discarded. Finally, the cells were resuspended in 1 mL PBS and analyzed by a BD FACSAria™ III ow cytometry system (BD).

Statistical analysis
All statistics were analyzed by GraphPad Prism 6.0 software and the data was expressed as the "mean ± SEM" of at least three independent repeats. The statistical signi cance of difference was assessed by unpaired Student's t-test for two group comparisons and a one-way ANOVA for more than two groups. The difference was considered signi cant when the corresponding P value was less than 0.05 (*) or 0.01 (**).

miR-487b-3p acts as a candidate regulator in goat skeletal myogenesis
To address the function of goat miR-487b-3p, the expression pattern of miR-487b in different tissues was detected by RT-qPCR analysis. The data showed that miR-487b-3p was enriched in skeletal muscle compared with heart, liver, spleen, lung and kidney during lamb-stage and adult-stage (Fig. 1a, and b). Besides, we found a higher expression level in lamb goat than adult goat (Fig. 1c). Signi cant upregulation of miR-487b-3p during myoblast proliferation was further observed (Fig. 1d). Bioinformatic analysis found that miR-487b-3p is located on Chromosome 21 and is composed of 22 nucleotides. The mature miR-487b-3p sequence is highly conserved among several species, including Ovis aries, Bos taurus, Mus musculus, Sus scrofa and Homo sapiens, according to the miRBase database (www.mirbase.org/) and UCSC (http://genome.ucsc.edu/) (Fig. 1e). These results suggested that miR-487b-3p might play a critical role in goat myogenesis.

RPG-based screening for genetically targeted goat myoblast cells
Three sgRNAs were designed to target the genome sequence of miR-487b-3p. The location of the three sgRNAs (sg1, sg2, sg3) is shown in Figure 2A with sg1 and sg2 targeting the seed sequence of miR-487b-3p. Then the three sgRNAs were cloned individually into the pX330 vector with Cas9 expression cassette (Fig. 2c) and were used to transfect goat myoblast cells. The frequencies of indels induced by the three sgRNAs were assessed using an in vitro digestion assay. The result suggested a much higher cleavage activity of sg2 with 12.5% indel frequency than sg1 and sg3 (Fig. 2b).
To enrich the genetically targeted goat myoblast cells by RPG surrogate reporter-based screening, we developed the RPG surrogate reporter system as we previously reported (Fig. 2c). After co-transfected with the CRISPR/Cas9 and RPG surrogate reporter vectors, the primary goat myoblast cells were subject to puromycin selection with the minimal lethal dose 0.75 µg/mL (Suppl. Figure 1). Further genotyping analysis of the screened cells by the digestion assay demonstrated 8.3%, 23.3% and 8.9% indel frequencies for sg1, sg2 and sg3, respectively (Fig. 2d). The genomic indels with random sizes adjacent to PAM sequence from the sg2 experiment group were further con rmed by TA cloning and subsequent sequencing (Fig. 2e). These results indicated that our RPG surrogate reporter system can be used for highly e cient enrichment of genetically targeted goat myoblast cells.

miR-487b-3p KO cell clones and off-target effect analyses
Several miR-487b-3p KO primary goat myoblast cell clones from the sg2 experiment group were obtained and expanded after the RPG surrogate reporter-based screening. The miR-487b-3p genomic loci of these cell clones were examined by PCR and sequencing. One of the cell clones with a deletion of 7 nucleotides (Fig. 3a) was chosen for off-target effect and further function analyses. T7E1 assay demonstrated that the indel frequency for expanded cell clone was 35.9 % (Fig. 3b). For off-target analysis, only 1 potential off-target site for sg2 was predicted by both the online design tool (http://crispr.mit.edu/) and Cas-OFFinder (Fig. 3c). The genomic locus of the off-target site was ampli ed by PCR and subjected to sequencing and T7E1 cleavage analyses (Fig. 3d). The sequencing result demonstrated no visible mutant peaks. On the other hand, T7E1 cleavage assay also showed no difference between the wild type and miR-487b-3p KO cells (Fig. 3e). In conclusion, these results suggested no detectable at the potential offtarget site in the miR-487b-3p KO cell clone, which could be used for further function analysis.
Knockout of miR-487b-3p promotes goat myoblast cells proliferation As for the function analysis, several assays were conducted to explore the effect of miR-487b-3p knockout on goat myoblast cells. Firstly, the barely detectable expression of miR-487b-3p in its KO cell clone was con rmed by RT-qPCR (Fig. 4a). Secondly, signi cantly up-regulated expression of cell cyclerelated genes, including PCNA, Cyclin E and CDK2, were detected in miR-487b-3p KO cells at both the mRNA (Fig. 4a) and protein levels (Fig. 4b). While the cell cycle inhibitor gene p27 was down-regulated in the opposite.
Next, the EdU incorporation assay demonstrated that the miR-487b-3p KO group had more labeled cells than that in the wildtype control group (Fig. 4c). The CCK-8 assay result also suggested that miR-487b-3p KO cells possess signi cantly higher viability compared with the wildtype control (Fig. 4d). What's more, the ow cytometric analysis result with more S-phage cells in the miR-487b-3p KO group (Fig. 4e) further revealed that the knockout of miR-487b-3p could promote the cell cycle process. Taken together, all these results indicated that the knockout of miR-487b-3p could promote the proliferation of goat myoblast cells.
IRS1 is a direct target of miR-487b-3p To explore the molecular mechanism of miR-487b-3p in goat myoblast, its potential target genes were predicted using the online miRBase and TargetScan software, and the IRS1 gene was chosen as the preferred candidate for further study (Fig. 5a). Strikingly, we found that miR-487b-3p and IRS1 had completely opposite expressing pro les during proliferating goat myoblast cells (Fig. 1d and 5b), and 100 nM miR-487b-3p mimics was enough to inhibit the IRS1 mRNA expression by 50% (Fig. 5c and d).
Next, the dual-luciferase reporter system was constructed to investigate whether the IRS1 gene is a direct target of miR-487b-3p. The 3'UTR fragments from IRS1 gene containing the predicted wildtype or mutant miR-487b-3p binding sites were cloned into the psi-CHECK-2 vector as shown in Figure 5A. As expected, the relative luciferase activity of the wildtype 3'UTR group was signi cantly inhibited as responded to miR-487b-3p mimics (P<0.05, Fig. 5e). In return, the IRS1 expression was signi cantly increased at both the mRNA and protein levels in miR-487b-3p KO cells ( Fig. 5f and g). Altogether, these results provide solid validation that IRS1 gene is a direct downstream target of miR-487b-3p in goat myoblast cells.
Knockout of miR-487b-3p affects myoblast proliferation by targeting IRS1 To further investigate the function of IRS1 gene in miR-487b-3p KO goat myoblast cells, the siRNA rescue experiment was conducted. miR-487b-3p KO goat myoblast cells were transfected with siIRS1 against IRS1 gene or the negative control siNC. The RT-qPCR and western blot detection results showed that the IRS1 mRNA and protein expression were both decreased by the transfection of siIRS1 ( Fig. 6a and b).
Further detection results demonstrated that the knockdown IRS1 gene by siIRS1 in the miR-487b-3p KO cells also caused altered expression of cell cycle-related genes. The expression of PNCA, Cyclin E and CDK2 genes were reduced, while the p27 gene increased, in the levels of both mRNA and protein ( Fig. 6c  and d). These results were just the opposite of those detected when no siRNA rescued as shown in Figure   4. We therefore could conclude that miR-487b-3p was a negative regulator to inhibit goat myoblast cells proliferation by targeting IRS1 gene.

Discussion
Recently, an increasing number of reports indicate that miRNAs play important roles in the regulation of animal myogenesis [10; 19; 28]. miR-487b-3p has been reported by previous study with high-throughput sequencing and RNAi to modulate myoblast cells proliferation in different species. This study was aimed to investigate the effect of miR-487b-3p on the cell proliferation of primary goat myoblast. The expression pro le of miR-487b-3p in different lamb or adult goat tissues was rstly illustrated. Then, its association with the cell proliferation of goat myoblast was further veri ed. Bioinformatics analysis found that the seed sequence of goat miR-487b-3p is identical between Ovis aries, Bos taurus and other species, suggesting important biological role of miR-487b-3p in regulation of evolutionarily conservative genes.
As a simple but e cient site-directed genome editing technology, CRISPR/Cas9 system has been widely used for genome editing in multiple organisms . However, the low transfection e ciency of primary cells and the di culty to select positive cell clones have always been the challenges in animal gene editing research [32; 33]. To enrich the genetically targeted goat myoblast cells, the RPG surrogate reporter system was constructed as we previously reported. The gene targeting e ciency in primary goat myoblast cells was signi cantly improved, and miR-487b-3p KO cell clones were obtained successfully by our RPG surrogate reporter-based screening. One of the cell clones with a deletion of 7 nucleotides in miR-487b-3p seed sequence veri ed with no detectable off-target effect was chosen for further function analysis.
Previous research has reported that the knockdown of miR-487b-3p by RNAi could suppresses the proliferation and differentiation of C2C12 myoblast [16]. In this study, we succeeded with the knockout of miR-487b-3p in goat myoblast cells by introducing indels into its seed sequence using CRISPR/Cas9. Both the results of EdU and CCK-8 assays demonstrated that the knockout of miR-487b-3p could accelerate the cell proliferation of primary goat myoblast cells. Consistently, the cell cycle analysis showed the G1-phase and G2-phase miR-487b-3p KO cells were decreased compared with wildtype control, while the S-phase KO cells was increased. A variety of molecules have been recognized to mediate the cell cycle progression. PCNA, Cyclin E, CDK2 and p27 are important regulators of the G1-S transition, which function to modulate the activity of cyclin-dependent kinases [34; 35]. The expression of these cell cycle related-genes was signi cantly altered as anticipated at both the mRNA and protein levels in the miR-487b-3p KO goat myoblast cells. Altogether, these results point to an essential role of miR-487b-3p in regulating the cell proliferation of goat myoblast cells.
IRS1 is the most important representative of the IRS protein family and the critical factor in insulin/IGF1signaling pathway [36; 37]. Here, we identi ed IRS1 as the target gene of miR-487b-3p in goat myoblast cells by bioinformatics analysis, RT-qPCR, western blot and dual-luciferase reporter assays. The IRS1 gene expression declined after the treatment with miR-487b-3p mimics and the expression pro le showed an inverse-correlation with that of miR-487b-3p during goat myoblast proliferation. It has been already acknowledged that IRS1 is a critical mediator of PI3K/Akt and AMPK signaling in the regulation of muscle growth and metabolism [38; 39]. As expected, the expression of IRS1 gene and the cell cyclerelated genes including PCNA, Cyclin E and CDK2 was signi cantly increased in the miR-487b-3p KO goat myoblast cells. On the contrary, siIRS1 could rescue the inhibition of IRS1 and these cell cycle-related genes in miR-487b-3p KO cells. These results suggested that miR-487b-3p targets the IRS1 gene directly at its 3'UTR, and the knockout of miR-487b-3p accelerates the cell proliferation of primary goat myoblast cells by relieving its inhibition of IRS1.

Conclusions
In summary, we succeed in obtaining the cell clones of miR-487b-3p KO primary goat myoblast by using CRISPR/Cas9 system and RPG surrogate reporter-based screening. miR-487b-3p targets IRS1 gene directly and the knockout of miR-487b-3p accelerates the cell proliferation of primary goat myoblast. These results provide novel insight into the function of miR-487b-3p in goat myoblast cells and will contribute to our understanding of the growth mechanism of goat skeletal muscle.
analyzed and interpreted the data; Ming Lyu., Kun Xu. and Wenqiang Zhang assisted data analysis and interpretation; Ming Lyu and Kun Xu wrote the manuscript. All authors approved the nal version of the manuscript.

Finding
This work was supported by grants from the China Postdoctoral Science Foundation (2018T111111 and 2015M580887), the National Natural Science Foundation of China (NSFC, 31702099).

Availability of data and materials
The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate All the animal procedures were raised in strict accordance with the guides of Northwest Agricultural and Forestry University (NWAFU) Animal Care and Use Committee. The experimental protocol was approved by the Departmental Animal Ethics Committee of Northwest A&F University (14-233, 10 December 2014).

Consent for publication
Not applicable.