Identification of the CAD gene from Eucalyptus urophylla GLU4 and its functional analysis in transgenic tobacco.

Cinnamyl alcohol dehydrogenase (CAD) catalyzes the final step in lignin biosynthesis. The genus Eucalyptus belongs to the family Myrtaceae, which is the main cultivated species in China. Eucalyptus urophylla GLU4 (GLU4) is widely grown in Guangxi. It is preferred for pulping because of its excellent cellulose content and fiber length. Based on GLU4 and CAD gene expression, a Eucalyptus variety low in lignin content should be obtained using transgenic technology, which could reduce the cost of pulp and improve the pulping rate, and have favorable prospects for application. However, the role and function of CAD in GLU4 is still unclear. In the present study, EuCAD was cloned from GLU4 and identified using bioinformatic tools. Subsequently, in order to evaluate its impact on lignin synthesis, a full-length EuCAD RNAi vector was constructed, and transgenic tobacco was obtained via Agrobacterium-mediated transformation. A significant decrease in CAD expression and lignin content in transgenic tobacco demonstrated a key role for EuCAD in lignin biosynthesis and established a regulatory role for RNAi. In our study, the direct molecular basis of EuCAD expression was determined, and the potential regulatory effects of this RNAi vector on lignin biosynthesis in E. urophylla GLU4 were demonstrated. Our results provide a theoretical basis for the study of lignin biosynthesis in Eucalyptus.

Genetic analysis of molecular markers for propamocarb residue in Cucumis sativus using quantitative trait locus mapping.

The use of pesticides to protect plants against harmful organisms, such as pathogenic microorganisms, is one of the most effective ways to improve agricultural production. However, the continuous use of pesticides might present a risk to human health, animals, and the environment. In this study, two cucumber (Cucumis sativus) varieties containing different levels of pesticide residues, D9320 and D0351, were selected to establish an F2 population. A genetic model and genetic linkage map were constructed. The results showed that the heredity of pesticide residues was dominated by an additive effect and was significantly influenced by non-additive factors in cucumber. QCp1 was detected as a quantitative trait locus (QTL) that might be involved in regulating the levels of pesticide residue in cucumber. Moreover, the cucumber genetic map was compared with the LG6 map, and the results indicated that this QTL was closely related to the level of pesticide residue in cucumber.

Association of vitamin D receptor gene polymorphisms with end-stage renal disease and the development of high-turnover renal osteodystrophy in a Chinese population.

Two single nucleotide polymorphisms (SNPs; TaqI and ApaI) in the vitamin D receptor (VDR) gene have been identified as risk factors for the progression of end-stage renal disease (ESRD). The purpose of our study was to confirm the reported association of these two SNPs with ESRD risk and progression of renal osteodystrophy in a Chinese Han population. A total of 452 ESRD patients and 904 matched-pair controls (based on age, gender, and body mass index) were included. Identification of VDR gene polymorphisms was performed using the polymerase chain reaction-restriction fragment length polymorphism method with TaqI and ApaI restriction enzymes. There was no association of the TaqI polymorphism with ESRD risk. However, significant associations were seen between ApaI (rs7975232) polymorphism and ESRD risk in the heterozygote model (AC/ AA; P = 0.002; OR = 1.4, 95%CI = 1.14-1.83), homozygote model (CC/AA; P = 0.007; OR = 1.8, 95%CI = 1.17-2.85) genotypes for rs7975232, allelic model (P < 0.001; OR = 1.4, 95%CI = 1.15-1.64), dominant model (P = 0.001; OR = 1.5, 95%CI = 1.19-1.87), and recessive model (P = 0.046; OR = 0.6, 95%CI = 0.42-1.00) between cases and healthy controls Moreover, we found a significant correlation between the genotype and allele distribution of ApaI and intact parathyroid hormone (iPTH) levels, where allele C carriers have increased iPTH levels. The ApaI polymorphism in the VDR gene appears to be a susceptibility locus for ESRD in Chinese individuals, and allele C carriers may have an increased risk of high-turnover renal osteodystrophy.

shRNA inhibits the expression of chicken telomerase reverse transcriptase in MDCC-MSB1 cells.

Here, we investigated the effects of blocking chicken telomerase reverse transcriptase (chTERT) in MDCC-MSB1 cells, using small-hairpin RNAs (shRNAs) to interfere with gene expression. shRNAs specific to chTERT mRNA were designed, cloned into DNA plasmid vectors, and transfected into MDCC-MSB1 cells. The transfected chTERT RNAs were expressed by the RNA polymerase machinery of the MDCC-MSB1 cells. mRNA expression in transfected MDCC-MSB1 cells was detected using real-time PCR. After transfection, telomerase activity was monitored via a modified telomeric repeat amplification protocol assay, and cell cycle analysis was performed using a flow cytometer. At 72 h after transfection, chTERT expression was considerably reduced in cells transfected with shRNA; the highest inhibition rate was 89%. Compared with the control group, telomerase activity was significantly reduced and the cells failed to progress to S phase. shRNA effectively reduced telomerase activity and prohibited the transition of MDCC-MSB1 cells from G2/M to S phase.

Identification of hub genes and pathways associated with retinoblastoma based on co-expression network analysis.

The objective of this paper was to identify hub genes and pathways associated with retinoblastoma using centrality analysis of the co-expression network and pathway-enrichment analysis. The co-expression network of retinoblastoma was constructed by weighted gene co-expression network analysis (WGCNA) based on differentially expressed (DE) genes, and clusters were obtained through the molecular complex detection (MCODE) algorithm. Degree centrality analysis of the co-expression network was performed to explore hub genes present in retinoblastoma. Pathway-enrichment analysis was performed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Validation of hub gene expression in retinoblastoma was performed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. The co-expression network based on 221 DE genes between retinoblastoma and normal controls consisted of 210 nodes and 3965 edges, and 5 clusters of the network were evaluated. By assessing the centrality analysis of the co-expression network, 21 hub genes were identified, such as SNORD115-41, RASSF2, and SNORD115-44. According to RT-PCR analysis, 16 of the 21 hub genes were differently expressed, including RASSF2 and CDCA7, and 5 were not differently expressed in retinoblastoma compared to normal controls. Pathway analysis showed that genes in 2 clusters were enriched in 3 pathways: purine metabolism, p53 signaling pathway, and melanogenesis. In this study, we successfully identified 16 hub genes and 3 pathways associated with retinoblastoma, which may be potential biomarkers for early detection and therapy for retinoblastoma.

Inheritance and quantitative trait locus analysis of low-light tolerance in cucumber (Cucumis sativus L.).

The low-light tolerance index was investigated in a set of 123 F2:3 lines during the seedling stage across 2 seasons, and the heredity of low-light tolerance was assessed via different ge-netic analysis methods. The results of the classical analysis showed that low-light tolerance is controlled by an additive-dominant poly-gene, and the polygenic inheritance rate of separate generations was >30%. In addition, 5 quantitative trait loci (QTLs) exhibited a low-light tolerance index across both seasons, including 2 QTLs (Llti1.1 and Llti1.2) on the 1st linkage group (variances of 6.0 and 9.5%) and 3 QTLs (Llti2.1, Llti2.1, and Llti2.1) on the 2nd linkage group (variances of 10.1-14.0%). The classical analysis method and QTL information on the heredity of low-light tolerance showed that it is controlled by several major genes and a mini-polygene. The results will facilitate the breeding of resistance to low-light stress in cucumber.

Relevance of E-cadherin expression to EGFR-TKI molecular targeted therapy sensitivity/resistance and its clinical significance.

We examined the effect of E-cadherin expression on epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) molecular targeted therapy sensitivity/resistance. We treated MCF-7, MDA-MB-231, T24, SiHa, H460, SK-HEP-1, MHCC97-H, and THP-1 cells with the EGFR-TKIs PD153035 and gefitinib, and then tested the drug-resistance and sensitivity using the MTT method, calculated IC50 values for each cell line, and compared the results to E-cadherin content. The MTT assay was used to determine the survival rates of MCF-7, MDA-MB-231, T24, SiHa, H460, SK-HEP-1, MHCC97-H, and THP-1 cells upon the action of EGFR-TKI (PD153035, gefitinib). For PD153035, the IC50 in MCF-7, MDA-MB-231, T24, and SiHa cells differed from that of H460, SK-HEP-1, MHCC97-H, and THP-1 (P < 0.05). Following gefitinib treatment, the IC50 values of MCF-7, MDA-MB-231, T24, and SiHa cells differed from those of H460, SK-HEP-1, MHCC97-H, and THP-1 cells (P < 0.01). The survival rate of MCF-7, MDA-MB-231, T24, and SiHa cells clearly decreased with increasing drug concentration, indicating the cells were sensitive to the drugs and that E-cadherin expression was positive; however, H460, SK-HEP-1, MHCC97-H, and THP-1 cells showed no significant decreased with increasing drug concentration, indicating that they were resistant to the drugs and that E-cadherin expression was negative. The survival rate of epithelial tumor cells through the action of EGFR-TKI is related to E-cadherin expression. E-cadherin may play a significant role in the sensitivity regulation of EGFR molecular targeting treatment. E-cadherin may provide important clues for selecting proper EGFR-TKI molecular targeting treatment.

MicroRNA network analysis identifies key microRNAs and genes associated with precancerous lesions of gastric cancer.

To identify potential targets for the early treatment and prevention of gastric cancer, microRNA (miRNA) expression profiles of precancerous lesions of gastric cancer were investigated. The miRNA microarray dataset GSE24839 was downloaded from Gene Expression Omnibus (GEO) and included 10 Helicobacter pylori-related gastritis samples and 10 gastric intestinal metaplasia samples. Differentially expressed miRNAs (DEMs) were screened using the Student t-test; P < 0.05 was considered to be statistically significant. Co-expression networks of total miRNAs and DEMs were constructed based on the Pearson correlation coefficient for the two diseases. Target genes of the DEMs were retrieved using miRecords and pathway-enrichment analysis was performed using a hypergeometric test. A total of 20 DEMs were obtained for H. pylori-related gastritis and gastric intestinal metaplasia samples, including 12 up-regulated and 8 down-regulated miRNAs. The identified DEMs appear to play key roles in gastric cancer, as the average degree of the DEM sub-network was higher than that of the total miRNA co-expression network. Furthermore, target genes for 6 DEMs (hsa-miR-106b, hsa-miR-193a-3p, hsa-miR-204, hsa-miR-30e, hsa-miR-519d, and hsa-miR-524-5p) are in Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including signal transduction, cell growth and death, and transport and catabolism. Among the target genes, 5 (RAB22A, SOX4, IKZF2, PLAG1, and BTBD7) were of interest because they can be simultaneously regulated by several DEMs. These findings suggest that these genes may be targets for modulating gastric cancer progression.

Formation of and countermeasures for subacute coronary stent thrombosis in elderly diabetic patients.

The aim of this study was to evaluate and investigate the pathogenetic mechanism and countermeasures of subacute thrombosis (SAT) after coronary stenting in elderly diabetic patients. The clinical characteristics and pathogenetic mechanisms in 3 cases of SAT after stent implantations in elderly diabetic patients were retrospectively examined to determine the treatment strategies for SAT. Among 98 patients with diabetes who had coronary stents implanted or were >60 years of age, three (3.06%) had SAT. One case of SAT was diagnosed by angiography; coronary balloon dilatation, thrombolysis, and re-perfusion resulted in full recovery in this case. The second case involved potential SAT, and in the third case, SAT was not ruled out. Two cases were characteristic of ST-segment elevation myocardial infarction, and one case, in which SAT was not ruled out, resulted in sudden death. SAT within a stent may be related to intraoperative stent malapposition caused by a grade C lesion, age, diabetes, chronic total occlusion, or postoperative irregular administration of medication.