[Cloning and expression analysis of U6 promoters in Panax quinquefolius].

The U6 promoter is an important element driving sgRNA transcription in the CRISPR/Cas9 system. Seven PqU6 promo-ter sequences were cloned from the gDNA of Panax quinquefolium, and the transcriptional activation ability of the seven promoters was studied. In this study, seven PqU6 promoter sequences with a length of about 1 300 bp were cloned from the adventitious roots of P. quinquefolium cultivated for 5 weeks. Bioinformatics tools were used to analyze the sequence characteristics of PqU6 promoters, and the fusion expression vectors of GUS gene driven by PqU6-P were constructed. Tobacco leaves were transformed by Agrobacterium tumefaciens-mediated method for activity detection. The seven PqU6 promoters were truncated from the 5'-end to reach 283, 287, 279, 289, 295, 289, and 283 bp, respectively. The vectors for detection of promoter activity were constructed with GUS as a reported gene and used to transform P. quinquefolium callus and tobacco leaves. The results showed that seven PqU6 promoter sequences(PqU6-1P to PqU6-7P) were cloned from the gDNA of P. quinquefolium, with the length ranged from 1 246 bp to 1 308 bp. Sequence comparison results showed that the seven PqU6 promoter sequences and the AtU6-P promoter all had USE and TATA boxes, which are essential elements affecting the transcriptional activity of the U6 promoter. The results of GUS staining and enzyme activity test showed that all the seven PqU6 promoters had transcriptional activity. The PqU6-7P with a length of 1 269 bp had the highest transcriptional activity, 1.31 times that of the positive control P-35S. When the seven PqU6 promoters were truncated from the 5'-end(PqU6-1PA to PqU6-7PA), their transcriptional activities were different in tobacco leaves and P. quinquefolium callus. The transcriptional activity of PqU6-7PA promoter(283 bp) was 1.59 times that of AtU6-P promoter(292 bp) when the recipient material was P. quinquefolium callus. The findings provide more ideal endogenous U6 promoters for CRISPR/Cas9 technology in ginseng and other medicinal plants.

Deficient and excess dietary selenium levels affect growth performance, blood cells apoptosis and liver HSP70 expression in juvenile yellow catfish Pelteobagrus fulvidraco.

We investigated the effects of deficient and excess dietary selenium (Se) on growth, blood cells apoptosis and liver heat shock protein 70 (HSP70) expression in juvenile yellow catfish (Pelteobagrus fulvidraco). After 8 weeks, yellow catfish (initial weight: 2.12 ± 0.01 g) fed isonitrogenous and isolipid diets containing <0.05 (deficient dietary Se) or 6.5 (excess dietary Se) mg Se/kg displayed a significantly lower weight gain ratio (WGR) than those fed a diet containing 0.23 (normal dietary Se) mg Se/kg. As dietary Se levels increased, liver Se concentration, glutathione peroxidase activity and the hepatosomatic index increased significantly. Plasma glucose concentration was highest in the normal treatment compared with the excess dietary Se treatment. Both deficient and excess dietary Se lead to increased reactive oxygen species (ROS) production and apoptosis ratio in blood cells, whereas only excess dietary Se increased their cytoplasmic free-Ca(2+) (CF-Ca(2+)) concentration. Excess dietary Se also resulted in the highest level of HSP70 expression, thereby possibly providing a protective mechanism against oxidative stress. These results indicate that both deficient and excess dietary Se restrained the growth of juvenile yellow catfish and caused oxidative stress. The overproduction of ROS may act as a signal molecule mediate apoptosis when dietary Se deficiency. Both ROS and CF-Ca(2+) were recorded when dietary Se excess, suggesting that Ca(2+) may be activated by Se and play a major role during Se-induced oxidative stress and cell apoptosis.

Female specific association between NNMT gene and schizophrenia in a Han Chinese population.

Accumulating evidence has shown that alterations in one carbon metabolism might play an important role in the pathogenesis of schizophrenia (SZ). Nicotinamide-N-methyltransferase (NNMT) is one of the key enzymes of one-carbon metabolism. To examine whether NNMT gene was associated with SZ in Han Chinese population, we selected seven single nucleotide polymorphisms (SNPs) in NNMT gene, and investigated its association with SZ from a cohort of 42 SZ patients and 86 healthy controls by Mass-ARRAY technology. Statistical analyses revealed that one (rs694539) of the SNPs in the female subgroup showed significant difference between SZ patients and controls both in genotypic (p= 0.0170) and allelic frequencies (p = 0.0059). We also found that the frequency of haplotype 'A G G C T C T' in the female patients was significantly higher than in controls (p=0.0015). Our results suggest that NNMT rs694539 may have a role in the etiology of SZ in a Han Chinese female population.

Inflammatory factors gene polymorphism in recurrent oral ulceration.

BACKGROUND: Some inflammatory factors play an important role in recurrent oral ulceration (ROU). The genetics mechanism of expression level of inflammatory factors is not clear in ROU, but from genetics the expression level of inflammatory factors at least partly depend on the gene polymorphisms. Therfore, we decided to investigate inflammatory factors gene polymorphism and its association with the susceptibility of recurrent oral ulceration in Chinese. METHODS: Genomic DNA was obtained from 42 subjects with recurrent oral ulceration, 86 subjects of healthy control individuals.Genotypes and alleles of 10 genes and 17 polymorphisms sites were analyzed by Mass-ARRAY Analyzer method. Then, the differences in distribution of each genotype and allele were compared. RESULTS: The statistical differences in distribution of TNF-α (rs1800629 and rs1800630) genotype and allele were observed among the groups with recurrent oral ulceration and healthy control individuals (P < 0.01), while VEGFA (rs1570360, rs833061, and rs2010963), EGF (rs4444903), TNF (rs361525), IL10 (rs1800896, rs1800872), IL2 (rs2069762), IL4 (rs2243250), Fas (rs1800682, rs2234767), IL12A (rs2243115, rs568408), IL12B (rs3212227), and IFNG (rs2430561) showed no statistical differences of genotype and allele in controls as compared to those in patients. CONCLUSIONS: This study suggests that the TNF-α (rs1800629 and rs1800630) genotype is an indicator for the susceptibility of recurrent oral ulceration.

MicroRNA-10b promotes migration and invasion through CADM1 in human hepatocellular carcinoma cells.

MicroRNA-10b (miR-10b) was recently reported to be dysregulated in some types of cancer and to play a role in invasion and metastasis. However, effects and potential mechanisms of action of miR-10b in the metastasis of hepatocellular carcinoma (HCC) have not been explored. In this study, we confirmed that miR-10b is highly expressed in metastatic HCC tissues and in metastatic HCC cell lines by qRT-PCR. Moreover, patients with higher miR-10b expression had significantly poorer overall survival, and high miR-10b expression was an independent predictor of poor prognosis. Inhibition of miR-10b reduced cell migration and invasion in MHCC97H cells, whereas over-expression of miR-10b in HepG2 cells increased cell migration and invasion. Bioinformatics and luciferase reporter assays revealed that miR-10b binds the 3'-UTR of CADM1 mRNA and represses its translation. Western blot and qRT-PCR showed that CADM1 is inhibited by miR-10b over-expression. Silencing of CADM1 resulted in substantially increased cell motility and invasion similar to that observed with over-expression of miR-10b in HepG2 cells. These results suggest that miR-10b may positively regulate the invasion and metastasis of HCC through targeting CADM1.

MiRNA-140 is a negative feedback regulator of MMP-13 in IL-1ß-stimulated human articular chondrocyte C28/I2 cells.

OBJECTIVE: Osteoarthritis is a degenerative joint disease, in which matrix metalloproteinase (MMP)-13 plays an important role. This study aimed to investigate miRNA-140-mediated negative regulation of MMP-13 expression in interleukin-1ß (IL-1ß)-stimulated human cartilage cells. METHODS: The human cartilage cell line C28/I2 was cultured in the presence of IL-1ß to mimic an osteoarthritic environment. Expression of miRNA-140 and MMP-13 was analyzed after 48 h by real-time RT-PCR and western blot analyses. MiRNA-140 mediated regulation of MMP-13 expression was analyzed by luciferase reporter assays and anti-miRNA-140 oligonucleotide transfection. Furthermore, miRNA-140 and MMP-13 expression was analyzed following DHMEQ treatment. RESULTS: Expression of miRNA-140 and MMP-13 was elevated in IL-1ß-stimulated C28/I2 cells. Bioinformatic prediction showed that the 3'-UTR of MMP-13 mRNA contained a potential binding miRNA-140 site and luciferase mRNA fused with 3'-UTR of MMP-13 mRNA was shown to be repressed by miRNA-140 in reporter assays. Expression of MMP-13 was elevated in IL-1ß-stimulated C28/I2 cells following anti-miRNA-140 oligonucleotide transfection. NF-κB activity was inhibited in DHMEQ treated IL-1ß-stimulated C28/I2 cells and was associated with decreased miRNA-140 and MMP-13 expression. CONCLUSION: Expression of miRNA-140 and MMP-13 was induced by IL-1ß. Expression of miRNA-140 inhibited MMP-13 in C28/I2 cells. Expression of miRNA-140 and MMP-13 was shown to be NF-κB-dependent.

Progress in researches on the pharmaceutical mechanism and clinical application of Ginkgo Biloba extract on various kinds of diseases.

Progress made over the pharmaceutical mechanism and clinical application of Ginkgo Biloba extract (GBE) on various kinds of diseases were reviewed in this paper. The effective elements contained in GBE are mainly kinds of Ginkgo flavonoid and Ginkgolide, which have marked protective effects on cardio-cerebral vascular and central nerve systems. In clinical practice, it is applied mostly in treatment of cardio-cerebral vascular diseases. Also it shows apparent effects in the treatment processes of some other diseases as an adjuvant, and therefore, has been gradually accepted by the medical circle in the world, proving to be a medicine of wide prospect in development and application.

Mitochondrial haplogroups and hypervariable region polymorphisms in schizophrenia: a case-control study.

Previous studies have detected associations between mitochondrial haplogroups and schizophrenia (SZ). However, no study has examined the relationship between major mitochondrial DNA (mtDNA) haplogroups and SZ in the Chinese population. The aim of this study was to assess the association between mtDNA haplogroups and SZ genesis in the Chinese Han population. We used a case-control study and sequenced the mtDNA hypervariable regions (HVR1, HVR2, and HVR3) in the Han population. We analyzed mtDNA haplogroups and HVR polymorphisms in 298 SZ patients and 298 controls. The haplotypes were classified into 10 major haplogroups: A, B, CZ, D, F, G, M, N, N9a, and R. Statistical analysis revealed that only N9a showed a nominally significant association with protection from SZ [1.68% vs. 6.38%, p=0.004, OR=0.251 (0.092-0.680); after adjustment for age and sex: p=0.006, OR=0.246 (0.090-0.669)]. Three HVR polymorphisms were found to be nominally significantly different between subjects with SZ and controls, and all except one (m.204T>C) are linked to the N9a haplogroup. Our results indicate that mtDNA haplogroup N9a might be a protective factor for SZ.

Hypervariable region polymorphism of mtDNA of recurrent oral ulceration in Chinese.

BACKGROUND: MtDNA haplogroups could have important implication for understanding of the relationship between the mutations of the mitochondrial genome and diseases. Distribution of a variety of diseases among these haplogroups showed that some of the mitochondrial haplogroups are predisposed to disease. To examine the susceptibility of mtDNA haplogroups to ROU, we sequenced the mtDNA HV1, HV2 and HV3 in Chinese ROU. METHODOLOGY/PRINCIPAL FINDINGS: MtDNA haplogroups were analyzed in the 249 cases of ROU patients and the 237 cases of healthy controls respectively by means of primer extension analysis and DNA sequencing. Haplogroups G1 and H were found significantly more abundant in ROU patients than in healthy persons, while haplogroups D5 and R showed a trend toward a higher frequency in control as compared to those in patients. The distribution of C-stretch sequences polymorphism in mtDNA HV1, HV2 and HV3 regions was found in diversity. CONCLUSIONS/SIGNIFICANCE: For the first time, the relationship of mtDNA haplogroups and ROU in Chinese was investigated. Our results indicated that mtDNA haplogroups G1 and H might constitute a risk factor for ROU, which possibly increasing the susceptibility of ROU. Meanwhile, haplogroups D5 and R were indicated as protective factors for ROU. The polymorphisms of C-stretch sequences might being unstable and influence the mtDNA replication fidelity.