Antiproliferative effects of isoalantolactone in human liver cancer cells are mediated through caspase-dependent apoptosis, ROS generation, suppression of cell migration and invasion and targeting Ras/Raf/MEK signalling pathway.

The main objective of this study was to evaluate the in vitro antiproliferative effects of isoalantolactone against liver cancer cells (Hep-G2) and also monitor its mechanism of action. The MTT assay was involved in proliferation assessments and phase contrast microscopy was used to check cellular morphology. Acridine orange/ethidium bromide staining along with western blotting was used to evaluate proapoptotic effects of isoalantolactone. DCFH-DA staining was used in ROS measurements. Transwell migration and invasion assay were executed to check the effects of isoalantolactone on migration and invasion of Hep-G2 cells. Western blotting was used to check the expressions of Ras/Raf/MEK signalling pathway in Hep-G2 cells. Results demonstrated that isoalantolactone significantly (*p<0.05 and **p<0.01) inhibited the proliferation of Hep-G2 cells in a concentration and time-reliant fashion. The IC50 value of the tested isoalantolactone molecule was found to be 71.2 µM and 53.4 µM at 12 h and 24 h time intervals respectively. Moreover, the antiproliferative effects of isoalantolactone were mediated through induction of caspase-dependent apoptosis and oxidative stress (ROS mediated). The proapoptotic effects of isoalantolactone were evident from morphological assessments and improved expressions of caspase-3, -8, and -9 and Bax while antiapoptotic Bcl-2 was reduced significantly. Additionally, antiproliferative and proapoptotic effects of isoalantolactone were found to be a consequence of blocking of Ras/Raf/MEK signalling in Hep-G2 cells. Furthermore, isoalantolactone significantly (*p<0.05) targeted the migration and invasion of Hep-G2 cells. In conclusion, these results validated that isoalantolactone shows strong antiproliferative activity against Hep-G2 liver cancer cells. Therefore, it could prove as a leading candidate in liver cancer research, drug discovery and design.

Estimation of genomic inbreeding coefficients based on high-density SNP markers in Chinese Holstein cattle.

In livestock, inbreeding coefficient based on pedigree information is usually used to evaluate the level of inbreeding. Recently, with cost reduction of high-density SNP genotyping, it's possible to analyze real genomic inbreeding degree using genomic information. In this study, utilizing high-density SNP chip data, we analyzed the frequency and distribution of runs of homozygosity (ROH) in 2107 Chinese Holstein cattle in Beijing area, and calculated 2 genomic inbreeding coefficients, i.e., 1) the proportion of ROH length in the total length of autosomal genome (Froh), and 2) the percentage of homozygous SNPs (Fhom). Then we analyzed the correlation between 2 genomic inbreeding coefficients and the correlation between genomic and pedigree inbreeding coefficients. We totally detected 44 676 ROHs that mainly ranged from 1 to 10 Mb. Various lengths of ROHs existed in the genome. There were more short ROHs than long ROHs. ROHs aren't evenly distributed in chromosomes. The area with most ROHs is in the middle part of chromosome 10. Strong correlation (r > 0.90) existed between 2 kinds of genomic inbreeding coefficients, but the correlation between pedigree and genomic inbreeding coefficients were much lower (r < 0.50). Our finding suggests that pedigree completeness influences the correlation between genomic and pedigree inbreeding. Genomic inbreeding measures may reflect individuals' real inbreeding, which could be a useful tool to evaluate population inbreeding.

Validation of PDE9A Gene Identified in GWAS Showing Strong Association with Milk Production Traits in Chinese Holstein.

Phosphodiesterase9A (PDE9A) is a cyclic guanosine monophosphate (cGMP)-specific enzyme widely expressed among the tissues, which is important in activating cGMP-dependent signaling pathways. In our previous genome-wide association study, a single nucleotide polymorphism (SNP) (BTA-55340-no-rs(b)) located in the intron 14 of PDE9A, was found to be significantly associated with protein yield. In addition, we found that PDE9A was highly expressed in mammary gland by analyzing its mRNA expression in different tissues. The objectives of this study were to identify genetic polymorphisms of PDE9A and to determine the effects of these variants on milk production traits in dairy cattle. DNA sequencing identified 11 single nucleotide polymorphisms (SNPs) and six SNPs in 5' regulatory region were genotyped to test for the subsequent association analyses. After Bonferroni correction for multiple testing, all these identified SNPs were statistically significant for one or more milk production traits (p < 0.0001~0.0077). Interestingly, haplotype-based association analysis revealed similar effects on milk production traits (p < 0.01). In follow-up RNA expression analyses, two SNPs (c.-1376 G>A, c.-724 A>G) were involved in the regulation of gene expression. Consequently, our findings provide confirmatory evidences for associations of PDE9A variants with milk production traits and these identified SNPs may serve as genetic markers to accelerate Chinese Holstein breeding program.

[Effects of annealing temperature on the structure and optical properties of ZnMgO films prepared by atom layer deposition].

In the present paper, we report the research on the effects of annealing temperature on the crystal quality and optical properties of ZnMgO films deposited by atom layer deposition(ALD). ZnMgO films were prepared on quartz substrates by ALD and then some of the samples were treated in air ambient at different annealing temperature. The effects of annealing temperature on the crystal quality and optical properties of ZnMgO films were characterized by X-ray diffraction (XRD), photoluminescence (PL) and ultraviolet-visible (UV-Vis) absorption spectra. The XRD results showed that the crystal quality of ZnMgO films was significantly improved when the annealing temperature was 600 degrees C, meanwhile the intensity of(100) diffraction peak was the strongest. Combination of PL and UV-Vis absorption measurements showed that it can strongly promote the Mg content increasing in ZnMgO films and increase the band gap of films. So the results illustrate that suitable annealing temperature can effectively improve the crystal quality and optical properties of ZnMgO films.

Low-temperature precipitation synthesis of flower-like ZnO with lignin amine and its optical properties.

A facile precipitation method has been developed to synthesize ZnO with [bis(2-aminoethyl)amino]methyl lignin (lignin amine) that is chemically modified from low-cost pulp industrial lignin. The obtained ZnO crystallites have been characterized to exhibit a hexagonal wurtzite structure, and their sizes have been determined at ca. 24 nm (mean value). These ZnO nanocrystallites are of high purity and well crystallized. Our present synthetic approach apparently exempts the commonly used calcining purification procedure. It is found that the morphology of ZnO and its specific surface area are capable of being tuned by varying the added lignin amine amount. Using the optimal 10 mL lignin amine, the synthesized ZnO exhibits flower-like morphology with proper specific surface area. Additionally, photoluminescence property of the obtainable ZnO displays two emissive bands at 383 nm (sharp) and in the range of 480 to 600 nm (broad) at room temperature. Their intensities were revealed to depend on the added lignin amine amount as well as on the molar ratio of Zn2+/OH-. The present investigation demonstrates that our method is simple, eco-friendly, and cost-effective for the synthesis of small-size ZnO materials.

[Advances on genome-wide association study for economically important traits in dairy cattle].

Milk performance traits have been considered as the most important production traits. With the development of balance breeding sense, much attention has been paid to the functional traits such as reproduction, type, health, and longevity traits, which is put into breeding programs. Identification of major genes or genetic markers for milk production and functional traits and their applications in breeding programs of dairy cattle are expected to improve the genetic progress of these traits. Occurrence of millions of SNPs in whole genome and high throughout genotyping techniques has made genome-wide association study to be an important strategy to identify genes responsible for economic traits in domestic animals. This paper reviewed the advances on genome-wide association study for milk production traits and functional traits in dairy cattle.

[Whole cDNA sequence cloning and expression of chicken L-FABP gene and its relationship with lipid deposition of hybrid chickens].

Liver fatty acid-binding protein (L-FABP) is closely related to intracellular transportation and deposition of lipids. A positive differential displayed fragment was found in the liver tissue among Silkie (CC), CAU-brown chicken (CD), and their reciprocal hybrids (CD and DC) at 8 weeks-old using differential display RT-PCR techniques (DDRT-PCR). Through recycling, sequencing, and alignment analysis, the fragment was identified as chicken liver fatty acid-binding protein gene (L-FABP, GenBank accession number AY321365). Reverse Northern dot blot and semi-quantitative RT-PCR revealed that the avian L-FABP gene was over-expressed in the liver tissue of the reciprocal hybrids (CD and DC) compared to their parental lines (CC and DD), which was consistent with the fact that higher abdomen fat weight and wider inter-muscular fat width observed in the reciprocal hybrids. Considering the higher expression of L-FABP may contribute to the increased lipid deposition in the hybrid chickens, the functional study of avian L-FABP is warranted in future.

[Analysis of DNA methylation in different chicken tissues with MSAP].

With methylation sensitive amplified polymorphism (MSAP), the DNA methylation levels and patterns of CCGG sites in genomes was analyzed among four different tissues and between parents and offsprings from three groups of adult chicken, White Leghorn, White Plymouth Rock, and their F1 hybrids. The results indicated that the degree of methylation was approximate 29.7% in muscle, 27.5% in liver, 27.5% in heart, and 26.1% in kidney. There was significantly different in the level of methylation in the 3 different groups and in 4 different tissues (P<0.05). The fully-methylated sites were less than the hemi-methylated sites among the 4 tissues, which was different from that of plants. The two tissue-specific MSAP fragments were isolated, sequenced, and characterized, both of which were located in the coding regions. These results clearly demonstrated that there was difference in the methylation level among various tissues and different groups, which suggested that the genetic factor may have effect on the individual methylation level.

[Identification of the complex vertebral malformation gene in Chinese Holstein and its association with dairy performance traits].

Complex vertebral malformation is caused by a single base mutation from G to T at the nucleotide position 559 in the bovine solute carrier family 35 member 3 (SLC35A3) gene exon 4 on Bos Taurus autosome (BTA) 3. The presence of the disease gene in Holstein dairy cattle has been reported in many countries. In this study, we examined 38 top Chinese Holstein sires in Beijing, four of which were identified as CVM carriers. Furthermore, 555 daughters of the four CV sires were examined and 44.0% of them were identified as heterozygotes at the mutation site. The association analysis between estimated breeding values (EBV) of dairy performance traits and the polymorphism showed that there were extremely significant differences between the carriers and the non-carriers (P<0.01). The EBVs of the five milk production traits of CVM carriers were significantly higher than those of non-carriers, and the lactation persistency and somatic cell score (SCS) were also higher in CVM carriers. Therefore, CVM gene seems to link with a QTL or gene associated with milk production traits on BTA3. It is recommended to cull the CVM carriers gradually for economical and breeding reasons.

Quantitative determination of acetylshikonin in macaque monkey blood by LC-ESI-MS/MS after precolumn derivatization with 2-mercaptoethanol and its application in pharmacokinetic study.

AIM: To develop and validate a novel precolumn derivatization method for the quantitative determination and pharmacokinetic application of acetylshikonin in macaque monkeys by liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). METHODS: 2-Mercaptoethanol was added to the blood sample as the derivatization reagent. The derivatization reaction formed 1 major derivation product, which was well correlated with acetylshikonin. The acetylshikonin concentrations in the biological samples were calculated by quantitative determination of the major derivation product using LC-ESI- MS/MS. Separation was achieved using a C18 column (2 mm x 50 mm, 5 microm) at room temperature and a linear gradient elution with a mobile phase containing methanol (1.96% acetic acid) and 10% methanol in water (1.96% acetic acid and 10 mmol/L ammonium acetate) at a flow rate of 0.2 mL/min. In addition, the major derivative, named derivative III, was identified by UV spectra, MS, and the (1)H-NMR and (13)C-NMR spectra. RESULTS: Good linearity was obtained within the range of 5 and 2000 ng/mL (r>0.99 using a linear regression model with 1/x2 weighting) for acetylshikonin. The interday and intraday precisions were found to be less than 12.3%, with the exception of the lowest concentration, which was less than 17.2%. The interday and intraday accuracies, which were between -3% and 0.6%, were also observed. After the administration of acetylshikonin (80 mg/kg, po) in macaque monkeys, the pharmacokinetic parameters were obtained through the non-compartmental analysis, where the area under the concentration-time curve to the last measurable concentration, the terminal elimination halflife, and the mean residual time were 615.4+/-206.5 ng x dh/mL,12.3+/-1.6 h, and 10.2+/-0.7 h, respectively. CONCLUSION: The method was validated and applied to the quantitative determination and pharmacokinetic study of acetylshikonin in the blood samples of macaque monkeys.

[Optimized multiplex PCR sets and genetic polymorphism of 30 microsatellite loci in domestic buffalo].

Nine multiplex-PCR-sets, including four triplex and five duplex combinations, were developed from 30 domestic buffalo microsatellite markers based on electrophoresis analysis of PCR products in denaturing polyacrylamide gels and visualization by silver-staining. Further, genetic variability on these microsatellites was examined in Xinglong buffalo of Hainan Province. Twenty-six loci exhibited genetic variation while four microsatellites (CSSM045, ILSTS008, RM099 and HMH1R) were monomorphism. The mean of number of alleles and the expected heterozygosity across 30 microsatellite loci were 4.7 and 0.534, respectively. These optimized multiplex-PCR-sets provide a technical basis for genetic diversity study and parentage test of domestic buffalo.

[Sequences and their polymorphisms of swine MHC-DQB, DRB proximal promoter region].

The primers, RF/RR and QF1/QR,were designed based on the homologous region of swine MHC-DQB and DRB exon1 sequences and HLA-DQB and HLA-DRB sequences. The 5' proximal promoter region (URR) nucleotide sequences of swine MHC-DQB and DRB were firstly obtained by PCR, cloning and sequencing techniques. There exist highly conserved sequence motifs including W, X, Y, CCAAT, and TATA-like boxes in the obtained sequences,showing the same organization of the conserved regulatory elements as in other species. 12 DRB-URR and 14 DQB-URR alleles were found by PCR-SSCP in 313 pigs. The aligned results of these alleles showed that there exist abundant polymorphism sites in both Porcine MHC-DQB and DRB proximal promoter region.

Study on the polymorphism of bovine lactoferrin gene and its relationship with mastitis.

Mastitis is a major cause of economic loss to the dairy industry. Lactoferrin (Lf) is known to contribute to resistance against bacterial infections. Hence, we decided to characterize the relevance between mastitis resistance and the variants of Lf gene. By using PCR-SSCP, five fragments within 5' region and all exons of bovine lactoferrin gene were amplified and identified the nucleotide diversity. For the five segments within the 5'-region: Lf5'-1, Lf5'-2, Lf5'-3, Lf5'-4, and Lf5'-5 from upstream to downstream, we found that three had base variation. Totally, mutations were observed in Lf5'-1, Lf5'-3, and Lf5'-5, exons 4, 8, 9, 11, 15, and intron 4. We analyzed the effects of all mutated loci on milk production traits with least squares method.

[Gene differential expression of liver tissues in crossbred versus purebred chicken and their relationship with heterosis of meat trait].

The concept of heterosis has already been put forward for a century. The hypothesis of Dominance, Superdominace and Epistasis has also been brought forward to explain the phenomenon of heterosis. As we know, there is spatio-temporal speciality about the expression of gene and only expressed genes contribute to the formation of heterosis. So the study on heterosis in expression level becomes more meaningful. A lot of studies on heterosis in this level have been done in plants, but there is no such study carried on animals in this area. In this study, the technique of mRNA Reverse Transcription Differential Display was used to research the heterosis molecular mechanism of animal. In order to expound the molecular genetic mechanism of animals heterosis, the 4 x 4 completely diallele cross experiment of 4 purebreds chicken was conducted among White Polymouth Rock (EE), Chinese Silk Chicken (CC), CAU Brown (DD) and White Leghorn (AA). The chicken of 16 cross combinations were reared to 8 weeks old, then 30 chicken in each combination were selected randomly and slaughtered. The traits of body weight of 8 weeks, wing weight, eviscerated weight, eviscerated weight with giblet, breast muscle yield, leg muscle yield, body length, abdomen fat weight, intramuscular fat width, tibia length were measured, and in which 8 individuals in each combination were selected randomly to collect the liver tissue samples, which were stored in liquid nitrogen or at -80 degrees C to be used for total RNA (TRNA) extracting. After the total RNA (TRNA) was extracted, 16 TRNA pools were formed in the same quantitative according to the concentration of 8 individual TRNA. They were reversely transcribed with three anchor primers H-T11 A, H-T11 G and H-T11 C. Then the reverse transcription PCR for each transcript product was done in two repeats at the same time with the same anchor primers and 8 random primers. The polyacrylamide gel electrophoresis of each PCR product was run in Bio-Rad Power 3,000 temperature control system. After electrophoresis, the gel was stained by AgNO3 according to the stain method described by Echt et al. The differential display bands in the polyacrylamide gel were counted. The band displayed is counted as 1 whereas no band is counted as 0 and only expression bands reproducible in two repeats were statistically analyzed. The correlation analysis between heterosis percentage and gene expression patterns was done with statistic analysis software (SAS) package. The statistic results indicated that among 690 total numbers of bands, the percentage of differential expression bands reproducible (457) is 66.23%. Eight kinds of gene differential expression patterns were found and listed as follow: 1): Band presents only in one purebred (P1);2): Band in one crossbred and its corresponding paternal purebred; or Band in one crossbred and its corresponding maternal purebred (P2);3): Band in purebreds and one crossbred (P3); 4): Band only in one crossbred (P4);5): Bands in both crossbreds and one purebred (P5);6): Bands only in both crossbreds (P6);7): Bands only in purebreds (P7);8): Bands both in purebreds and crossbreds P8. The differential expression of gene between purebred and crossbred chicken was detected for the first time. The proportion of each pattern in each kind of purebred combination is different. The percentage of P8 (75.34%) is the highest. The total percentage of differential expression patterns (24.66%) showed that the gene differential expression exists as a matter of fact. Among all the gene differential expression patterns, the percentage of P3 is the highest whereas the percentage of P7, P6 and P4 is very low, it indicated that different genes may have different expression patterns in purebreds and crossbreds. The results are similar to the study results on plants, which indicates that the gene differential expression between purebred and crossbred exists universally in biology. The correlation between gene expression patterns and heterosis percentage was studied, but correlation between P8 and the heterosis percentage is not significant (P > 0.05), it indicates that some patterns of gene differential expression may be the molecular genetic basic of heterosis. Among all the gene differential expression patterns, each pattern affects the expression of meat trait in different manner. There is significantly negative correlation between P4 and heterosis percentage of body weight of 8 weeks, breast muscle yield, leg muscle yield, eviscerated weight with giblet and eviscerated weight (P < 0.05); P1 is of significantly negative correlation with heterosis percentage of abdomen fat weight (P < 0.05) and of very significantly negative correlation with heterosis percentage of body length (P < 0.01); The negative correlation between P2 and heterosis percentage of intramuscular fat width is significant (P < 0.05); The positive correlation between P7 and heterosis percentage of leg muscle yield, wing weight, eviscerated weight with giblet and intramuscular fat width is significant (P < 0.05); The positive correlation between P5 and heterosis percentage of tibia length (P < 0.05) is significant. These results show that these 5 kinds of patterns play important role in heterosis forming of meat trait. P1 and P7 show that expressed gene in purebreds is depressed; P4 indicates that new gene expression occurs in crossbreds; P5 reveals that expressed gene only in one purebred express in all crossbreds. All genes of crossbreds come from purebred, which are not only the simple adding of these purebred genes, giving birth to unknown interaction between these genes coming from different purebreds, then leading to differential expression of genes. These gene differential expressions maybe form the heterosis of meat trait.

[Polymorphism analysis of the GOLA-DRB3 gene digested with HaeIII in Mongolian goat and Kazakh goat].

The exon2 of GOLA-DRB3 gene was amplified and a uniform fragment of 285 bp was obtained in Mongolian Goat and Kazakh Goat. The 285bp PCR product was digested with restriction endomuclease HaeIII and genetic polymorphism was investigated by PCR-RFLP. Seventeen kinds of genotypes were found in two populations,which were controlled by seven alleles. There are significant differences in some genotypic frequencies and gene frequencies between the two populations (P<0.10, P<0.05, P<0.01); The results of chi(2) test showed that genotypes of GOLA-DRB3 gene in two populations did not fit with Hardy-Weinberg equilibrium (P<0.01).

[Differential expression of CIDH gene between purebreds and hybrids in chicken and relationship with heterosis].

The technique of silver staining mRNA Differential Display was used to check the molecular genetic mechanism of heterosis in chicken. A differentially expressed fragment CE15A15, which was only expressed in two crossbreds, was found between purebreds and crossbreds. It is proved that the fragment intensively expresses in crossbreds through reverse Northern dot blot and RT-PCR. It shares 97% of sequence identity with a partial cDNA sequence of chicken that has been deposited in GenBank (AW198493) and believed to be identity with the cytosolic isocitrate dehydrogenase (CIDH) gene (ID:AF048831) of Microtus mexicanus. This suggests that the identified fragment may be a partial cDNA sequence of CIDH gene. The statistic analyses of phenotypes showed that the body weight and the carcass traits in crossbred of Silk Chicken (CC) x White Polymouth Rock (EE) and White Polymouth Rock (EE) x Silk Chicken (CC) had negative heterosis, while their fatty traits had positive heterosis. CIDH directly participates in the biosynthesis of fatty acids. This indicates that differential expression of the fragment between purebreds and crossbreds might be closely related to heterosis of crossbreds.

[Analysis of the 5'-region of bovine lactoferrin gene].

To detect DNA variation of bovine lactoferrin gene, the sequence of 5'-regulatory region has been studied by PCR-SSCP. Among the five sub-sequences of this region, Blf 5'-1(227bp),Blf 5'-3(175bp) and Blf 5'-5(293bp)were found to be polymorphic. After sequencing the Blf 5'-1, G-->A and T-->G transitions were identified at the sites of -926 and -915 respectively. In the Blf 5'-3 fragment, a G insertion at -478 was found. Transversions of C-->A, G-->C at -28 and +33 respectively were also detected in the Blf 5'-5 region. The frequencies of mutated alleles were 0.101,0.112 and 0.237, respectively, in the three regions. By applying an analysis tool of TFSEARCH ver1.3, we studied the potential elements and protein factors which bind to the 5' -region, and discovered the different regulatory elements of pre-mutation and post-mutation.

[Polymorphisms of exon 2 of MHC-DRB3 gene in Mongolian and Kazakh Sheep].

MHC(major histocompatibility complex) is a chromosomal region consisting of a group of closely linked loci which are highly polymorphic, which plays a central role in the immune system of animals and is almost found in all vertebrates. Its expressional product is MHC antigen of which main function is antigen presentation. In this study, the exon 2 of MHC-DRB3 gene was amplified by PCR and a uniform fragment of 285 bp was obtained in Mongolian Sheep and Kazakh Sheep. The 285 bp PCR product was digested with restriction endomuclease HaeIII and genetic polymorphism was investigated by PCR-RFLP. Seven alleles were found: A(168 bp/117 bp), B(154 bp/117 bp/14 bp), C(154 bp/65 bp/52 bp/14 bp), D(168 bp/65 bp/52 bp), E(154 bp/131 bp), F(154 bp/66 bp/65 bp), H(220 bp/65 bp). There are seventeen genotypes in the tested populations, BB(154 bp/117 bp/14 bp), CC(154 bp/65 bp/52 bp/14 bp), EE(154 bp/131 bp), HH(220 bp/65 bp), FF(154 bp/66 bp/65 bp), AB(168 bp/154 bp/117 bp/14 bp), AC(168 bp/154 bp/117 bp/65 bp/52 bp/14 bp), AE(168 bp/154 bp/131 bp/117 bp), AH(220 bp/168 bp/117 bp/65 bp), BC(154 bp/117 bp/65 bp/52 bp/14 bp), CE(154 bp/131 bp/65 bp/52 bp/14 bp), CD(168 bp/154 bp/65 bp/52 bp/14 bp), CF(154 bp/66 bp/65 bp/52 bp/14 bp), CH(220 bp/154 bp/65 bp/52 bp/14 bp), DE(168 bp/154 bp/131 bp/65 bp/52 bp), EH(220 bp/154 bp/131 bp/65 bp), FH(220 bp/154 bp/66 bp/65 bp). By analyzing restriction map, polymorphic sites were detected at base position 154, 168 and 220. Statistical results indicated that there are significant differences of some genotypic frequencies and allelic frequencies between Mongolian Sheep and Kazakh Sheep(P < 0.10, P < 0.05 or P < 0.01). CC, FF, BC and B, C, F are favorable genotypes and favorable alleles respectively in Mongolian Sheep and Kazakh Sheep, In addition, HaeIII CE and HaeIII E is favorable genotype and favorable allele respectively in Kazakh Sheep. The results of chi 2 test showed that genotypes of MHC-DRB, gene in Mongolian Sheep and Kazakh Sheep did not fit with Hardy-Weinberg equilibrium(P < 0.01).