The leucoanthocyantin reducase (LAR) gene, an important functional gene of catechins biosynthesis pathway, was cloned from Fagopyrum dibotrys (D.Don) Hara by degenerate PCR and rapid amplification of cDNA ends (RACE). The full-length cDNA of FdLAR is 1 581 bp (GenBank accession: JN793953), containing a 1 176 bp ORF encoding a 391 amino acids protein, and its 3'-untranslated region has an obvious polyadenylation signal. The recombinant plasmid containing FdLAR completed ORF was transformed into E. coli BL21 (DE3). The target fusion peptide with molecular weight of 66 kD was expressed under the condition of 16 degrees C and induced by IPTG at final concentration of 1.0 mmol x L(-1). Bioinformation analysis indicated that the amino acid sequence of FdLAR showed great homology to other LAR with the NADB-Rossmann conversed domain in the N-terminus. Real-time quantitative PCR was used to detect the expression levels of FdLAR gene during different development periods. The determination of flavonoids contents in appropriate rhizomes showed that the relationship between FdLAR gene expression and the accumulation of flavonoids displayed different trends during vegetative growth and reproductive growth stages, suggesting that the FdLAR gene may be involved in the pathway of flavonoid metabolisms in Fagopyrum dibotrys.

Objective To discuss the role of platelet-actived factors and hepatic and renal function in the development of pre-eclampsia and eclampsia by detecting the levels of the GP Ⅱb/Ⅲa in the blood, Pt and hepatic and renal function of the patients with pre-eclampsia and eclampsia. Method GP Ⅱb/Ⅲa, Pt and hepatic and renal function were measured in the normal non-pregnancy women, normal late-pregnancy women and pre-eclampsia and eclampsia women. Results The level of GP Ⅱb/Ⅲa in patients with severe pre-eclampsia and eclampsia was higher than that in non-pregnancy, normal late-pregnancy and mild pre-eclampsia (P<0.01). The count of Pt in patients with severe pre-eclampsia and eclampsia was lower than that in non-pregnancy, normal late-pregnancy and mild pre-eclampsia (P<0.01). Except ALB, the hepatic and renal function had significant difference among severe pre-eclampsia and eclampsia, normal late-pregnancy and mild pre-eclampsia. Conclusion Detecting the GP Ⅱb/Ⅲa, Pt and hepatic and renal function have clinical significance in severe pre-eclampsia and eclampsia.

Samples of skeletal muscle in forearm were taken from 60 sites in each of 10 normal autopsy subjects (male 7, female 3) between 3 and 69 years of age. The distribution and composition of type I and type II fibers in the muscles of the forearm were studied by histochemical method for myofibrillar ATPase. The results showed that the mean percentage of type I fibers ranged between 45% to 59% in the muscles of the forearm. In general, the slow contracting fibers of the forearm muscles were slightly higer than 50%. In the flexors and extensors of the forearm the respective percentage of type I fibers were 49.8% and 55.3%. The slow contraction fibers of the extensors muscle group showed higher proportion of type I fibers than those of the flexor group and the difference is statistically significant (P

MicroRNAs (miRNAs or miRs) are a class of short, non-coding RNAs that participate in various oncological processes. This study aims to explore the roles of microRNA-34a (miR-34a) in invasive urothelial bladder carcinoma. miR-34a was transfected into bladder cancer cell lines 253J and J82. The miR-34a expression levels in tissues and cells were detected by using qRT-PCR. The Notch1 expression was detected by qRT-PCR and Western blotting. Cell migratory and invasive abilities were measured by Transwell chamber assay. Bioinformatics and luciferase assay were performed to predict and analyze the binding sites between miRNA-34a and Notch1. It was found that there was aberrant expression of miR-34a in bladder cancer tissues. Moreover, we revealed that ectopic expression of miR-34a suppressed cell migration and invasion, while forced expression of Notch1 increased cell migratory and invasive abilities. Finally, we observed that miR-34a transfection significantly down-regulated luciferase activity and reduced the mRNA and protein levels of Notch1. Our study concluded that microRNA-34a antagonizes Notch1 and inhibits cell migration and invasion of bladder cancer cells, which indicates the tumor-suppressive function of microRNA-34a in bladder cancer.

Objective To explore the effects of amniotic lacrimal duct stenting on the prevention of dry eye in castrated rabbits.Methods Thirtysix healthy male rabbits were selected,the third eyelid were cut off and antiinfection treatment were given,which were randomly divided into 3 groups (12 cases in each group),the castrated male rabbits models were made.Among them,group A was negative control group,group B was dry eye model group,group C was group of lacrimal amniotic membrane group.At 2 weeks before implantation of amniotic lacrimal duct stent,2 weeks,4 weeks and 6 weeks after implantation,the fluorescent (FL) examination,Western blot,Schirmer I examination,immunofluorescence staining and corneal confocal microscopy were performed.Results The levels of tear secretion and FL in the three groups among different time points were significantly different (F=7.126,P =0.009;F =9.658,P =0.016),and there were significant differences among three groups (F =12.582,P =0.005;F =13.187,P =0.013).The tendency of tear secretion and FL in the three groups were also significantly changed (F =8.531,P =0.007;F =10.652,P =0.019).The epithelial basal cells at 6 weeks after implantation in three groups were 3811 ±414,3820 ± 314,2789 ± 353,and the density of inflammatory cells was 266 ±28,266 ± 29,67 ± 13,there were significant differences among three groups (F =13.442,P =0.012;F =9.231,P =0.021).The K1 6 staining in the duct epithelium were negative,and the expression of α-SMA in the lacrimal duct tissue of group A,B and C was not changed at all time points after implantation of amniotic lacrimal stent,and there was no significant difference (F =14.681,P =0.002).Conclusion The amniotic lacrimal stent implantation has certain effect on the prevention of dry eye in rabbit.

There are some shortcomings of animal experiments applied in chemical toxicity testing, such as long period, large cost, species differences and dose differences, which limit the use of animal experiments' results in predicting human toxicity.Accordingly, we established a toxicity testing alternative screening system in line with the toxicity endpoints which required in chemical safety evaluation and risk assessment (genotoxicity, carcinogenicity, reproductive toxicity, acute toxicity and general toxicity) based on 3R principles (replacement, reduction, refinement) for animal experiments.This system covers most of the endpoints of toxicity assessment, and molecular biology technology was also applied to integrate the toxicity test, as well as some operation was optimized in order to shorten the experimental period, reduce experimental costs, improve animal welfare.Furthermore, the results from the screening system have higher clinical relevance because it is based on the toxicity mechanisms.

ObjectiveTo investigate the effects of over expression of miR-34a on cellular proliferation and migration in bladder cancer cell line J82 by targeting Notchl.MethodsmiR-34a was predicted as a putative gene which can target Notchl through bioinformatics analysis,qRT-PCR and Western blot were performed to measure the expression levels of Notchl and miR-34a in invasive transitional cell carcinoma of bladder (TCCB) tissues and J82 cells transfected with miR-34a.Luciferase assay was employed to determine if miR-34a could target Notchl through binding to the 3'-untranslated region (3'UTR) of Notchl mRNA.J82 cells were transfected with pcDNA3.0-miR-34a or pcDNA3.0 control plasmid.MTS colorimetry was used to evaluate the effect of miR-34a on cell proliferation.The effect of miR-34a on cell migration was assessed by transwell migration assay.ResultsThe expression level of miR-34 in invasive TCCB tissues was lower than in adjacent bladder tissues (0.016(0.018) vs 0.042 (0.059),N =16; P =0.0006).On the contrary,the average levels of Notchl mRNA and protein were higher in tumors than in adjacent bladder tissues (2.765(2.156) vs 2.312(1.365),N =16; P =0.0025 and 0.857 ±0.197 vs 0.648 ±0.171 ;P ＜0.0001 ).After the transfection of miR-34a,the expressive level of miR-34a in J82 was highly induced ( (2.408 ±0.789) × 10-4 vs(0.153 ±0.029) × 10-4; P =0.0026).However,the expressive levels of Notchl mRNA and protein were obviously decreased (3.001 ± 0.106 vs 4.998 ± 1.053 ; P =0.0308 and 0.747 ± 0.050 vs 0.988 ± 0.102 ; P =0.0215 ).The results of luciferase assay showed that firefly activity was highly dimished (0.422 ± 0.028 vs 2.392 ± 0.148 ; P ＜ 0.0001 ).Cellular proliferation was inhibited after the transfection of miR-34a in J82 (P ＜ 0.0001 ).Moreover,number of migration cells of J82 was significantly reduced after the ectopic expression of miR-34a ( 179.3 ± 21.02 vs 269.7 ± 23.71 ; P =0.0078 ).ConclusionsmiR-34a inhibits the cellular proliferation and migration of bladder cancer cell line J82 via binding to the 3UTR of Notchl mRNA.

Using RACE with a Fagopyrum dibotrys callus cDNA library, one clone, named FdMYBP1, encoding a putative R2R3 MYB protein was identified. FdMYBP1 appeared to be a full-length cDNA of 1159 bp encoding a protein of 265 amino acids. Through structure and property analysis of FdMYBPI with bioinformational methods, it was found that the amino acid sequence of FdMYBP1 showed great homology to other MYBP with the R2R3 repeat region in the N-terminus. Southern blot analysis indicated that FdMYBP1 belongs to a single copy gene in F. dibotrys genomes. The FdMYBP1 gene has the same classic characters with other MYBP and probably involved in the pathway of flavonoid metabolisms.
Cloning, Molecular ; Fagopyrum ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Molecular Sequence Data ; Plant Proteins ; genetics ; metabolism ; Proto-Oncogene Proteins c-myb ; genetics ; metabolism

Objective: To investigate the level of vitamin B12 in children with autism spectrum disorder(ASD), and provide a theoretical basis for early detection and drug treatment of ASD. Methods: A total of 89 ASD cases and 89 matched controls were collected. The levels of urinary methylmalonic acid (MMA) and serum vitamin B12, Transcobalamin Ⅱ (TCN2) were determined by enzyme-linked immunosorbent assay (ELISA). TCN2 gene rs1801198 was genotyped by SNaPshot. Results: The serum levels of vitamin B12 and TCN2 in children with ASD [(369.08±131.88)pmol/L, (1.56±0.16)ng/mL] were significantly lower than those in the control group[(485.16±200.33)pmol/L, (1.71±0.17)ng/mL](t=-5.47, -5.92, P<0.05). The level of MMA in urine of ASD children [(758.97±106.96) ng/mL] was significantly higher than that in the control group[(693.66±121.72)ng/mL](t=3.94, P<0.05); The genetic polymorphism of rs1801198 locus was not associated with the risk of ASD(P>0.05), and there was no significant correlation with serum TCN2 level(F=1.16, P>0.05). Conclusion ASD children are at a potential deficiency of vitamin B12 and should strengthen their nutritional interventions while conducting ASD interventions.

Objective: To explore the differences of temperament characteristics between children with autism spectrum disorder (ASD) and normal children, and to provide evidence for early detection of ASD children and the development of personalized treatment plans. Methods: In this case-control study, we enrolled 129 ASD children and 129 normal children aged 3-7 years. The Behavioral Style Questionnaire (BSQ) scale was used to assess the temperament. Results: ASD children got higher scores in terms of "activity level", "withdrawal", "adaptability", "emotional nature", "persistence", and "response threshold" temperament dimensionality scores(P<0.05), and lower scores in terms of "rhythmical", "response intensity" temperament dimensionality scores than normal children(P<0.05). However, there was no significant difference in "attention dispersiveness" between ASD group and control group(P>0.05). Among the children in the ASD and control group, the proportion of each temperament type was "easy to raise temperament type" (41.8% vs 62.8%), "partially easy to raise temperament type" (31.8% vs 27.9%), "partially difficult to raise temperament type" (17.1% vs 6.2%), “slow-up-towarm temperament type" (7.7% vs 2.3%) and "difficult to raise temperament type" (1.6% vs 0.8%).Statistical analysis showed that the rate of "easy to raise temperament type" was lower than that in normal children(P<0.05), while the rates of "partially easy to raise temperament type", "partially difficult to raise temperament type", "difficult to raise temperament type", and "slow-up-to-warm temperament type" in ASD children were higher(P<0.05). Conclusion There was significant difference in temperament characteristics between ASD children and normal children. The evaluation of temperament type contributes to early detection of ASD children and provides a reference for their behavioral correction.