Over-activation of EFTUD2 correlates with tumor propagation and poor survival outcomes in hepatocellular carcinoma.

PURPOSE: Elongation factor Tu GTP-binding domain containing 2 (EFTUD2) is an essential constituent of U5 small nuclear ribonucleoproteins (snRNPs) and plays a crucial role in spliceosome activation and cancer. The mechanism of EFTUD2 on carcinogenesis and development of liver cancer still need further study. METHODS: Bioinformatic analysis was performed to find differential expressed genes and related pathways. Western blotting and quantitative PCR assays were used to verify the EFTUD2 expression in HCC cell lines and tumor tissues of liver cancer patients. Transfection of shRNAs in SKHEP1 and Huh7 cell lines was conducted to explore the mechanisms of EFTUD2 in HCC. CCK-8 method, colony formation, and cell cycle detection kit were used to detect the proliferation. A tumor model in nude mice was used to explore the role of EFTUD2 in liver cancer in vivo. RESULTS: Based on the tumor tissues and para-tumor tissues in our HCC patients, we identified EFTUD2 as highly expressed in HCC tissues (P < 0.001). Bioinformatic analysis from the TCGA database also supported this biological phenomenon (P = 1.911e-17). Furtherly, the results of clinical specimens and TCGA data suggested that higher EFTUD2 expression levels correlated with high histologic grades, high pathological grades, and poor survival prognoses in HCC patients. And knockdown of EFTUD2 suppressed cell proliferation and colony formation in vitro. In vivo, knockdown of EFTUD2 constrained the tumor growing and expansion derived from SKHEP1 cells and induced a decrease in the tumor volume and tumor weight resected from nude mice. Furthermore, RNA sequencing based on EFTUD2 knockdown revealed that EFTUD2 affected target genes concerned with the cell cycle. Flow cytometric analyses in the SKHEP1 cell model revealed that knockdown significantly suppressed cell cycle course and caused cell cycle arrest in the G1 phase. CyclinD1 proteins were also inhibited by knocking down of EFTUD2. CONCLUSION: EFTUD2 is markedly overexpressed in HCC tumor tissues. High EFTUD2 expression in HCC patients is associated with clinical features. Moreover, we confirmed that EFTUD2 shows a pivotal role in HCC cell proliferation and cell cycle course and could be a possible therapeutic avenue in HCC through disturbing EFTUD2.

Karyotype analysis and ribosomal gene localization of spotted knifejaw Oplegnathus punctatus.

The spotted knifejaw, Oplegnathus punctatus, is an important aquaculture fish species in China. To better understand the chromosomal microstructure and the karyotypic origin of this species, cytogenetic analysis was performed using Giemsa staining to identify metaphase chromosomes, C-banding to detect C-positive heterochromatin, silver staining to identify the nucleolus organizer regions (Ag-NORs), and fluorescence in situ hybridization (FISH) for physical mapping of the major (18S rDNA) and minor (5S rDNA) ribosomal genes. The species showed a karyotype of 2n = 48 for females, composed of 2 submetacentric and 46 telocentric chromosomes, with a fundamental number (FN) = 50, while the karyotype of males was 2n = 47, composed of 1 exclusive large metacentric, 2 submetacentric, and 44 telocentric chromosomes, with FN = 50. These karyotype results suggest that O. punctatus might have an X1X1X2X2/X1X2Y multiple sex chromosome system. C-positive heterochromatin was distributed in the centromeres of all chromosomal pairs and in the terminal portions of some chromosomes. A single pair of Ag-positive NORs was found to be localized at the terminal regions of the short arms of the subtelocentric chromosome pair, which was supported by FISH of 18S rDNA. After FISH, 5S rDNA were located on the interstitial regions of the smallest telocentric chromosome pair. This study was the first to identify the karyotype of this species and will facilitate further research on karyotype evolution in the order Perciformes.

Novel polymorphic microsatellite markers for Bellamya and their application in population genetics of three species.

Bellamya is a widely distributed freshwater snail genus in China; however, its genetic diversity is completely unknown. Sixty-five novel microsatellite loci were isolated and characterized from a microsatellite-enriched library of Bellamya aeruginosa genomic DNA. Most of the 65 loci were successfully amplified. We found high polymorphic information content values for these loci, ranging from 0.235 to 0.892. There were 3 to 12 alleles per locus, and the HE and HO varied from 0.425 to 0.953 and 0.026 to 1.000, respectively. Fifteen loci deviated significantly from Hardy-Weinberg equilibrium after Bonferroni's correction. All 65 SSR markers were tested in an additional five Bellamya species, and 96.9% of the 325 locus/taxon combinations tested resulted in cross-species amplification. Seven polymorphic microsatellite markers were randomly selected for comparison among nine populations of three species. All populations had moderate to high genetic diversity. In genetic distance-based cluster analysis, the populations of B. aeruginosa and B. dispiralis formed species-based clusters, whereas populations of B. angularia did not. The three examined Bellamya species could be differentiated using SSR markers. These microsatellite loci should be useful for genetic diversity analysis, analysis of phylogenetic relationship, and species delimitation of Bellamya.

NEDD4-1 and PTEN expression in keloid scarring.

Keloid scarring remains a major problem in plastic surgery. The aim of this study was to determine the expression of the PTEN tumor suppressor and NEDD4-1 genes in keloid tissue and explore their effect on the formation of such scarring. Twenty keloid patients were enrolled in the study and underwent surgical removal of keloid tissue. No patient had received chemotherapy and/or radiotherapy prior to treatment. PTEN and NEDD4-1 mRNA expression was detected by reverse transcription PCR, while PTEN protein expression was assessed using immunohistochemistry. Our results showed that levels of PTEN were significantly diminished in keloid samples (P < 0.05), whereas those of NEDD4-1 did not significantly differ between keloid tissue and normal skin (P > 0.05). Furthermore, we found that NEDD4-1 expression is high and inversely correlated with that of PTEN in keloids. Our results suggest that the PTEN/PI3K/AKT pathway may play an important role in keloid formation and reduces PTEN expression in such tissue. Finally, although NEDD4-1 has previously been identified as a factor in keloid susceptibility, and the protein for which it encodes is known to degrade PTEN by catalyzing its polyubiquitylation, the detailed mechanism behind its involvement in keloid formation needs to be further studied.

Significance of detecting IgH and TCRγ gene rearrangements in patients with hemopoietic maligancies by real-time quantitative PCR.

The aim of our study was to investigate the association of IgH and TCRγ gene rearrangements in hematological malignancies with the disease and clinical application. IgH and TCRγ gene rearrangements were determined in 69 paraffin and bone marrow specimens with SYBR Green I fluorescent dye and RQ-PCR method, including 21 paraffin-embedded tissues of the onset cases and 48 bone marrow samples, representing 15 ALL and 25 AML cases. After chemotherapy, 8 cases were NHL; the 10 cases of the negative control group were healthy people. Among the ALL cases, the IgH rearrangement occurred in 80.0%, the TCRγ rearrangement in 46.7%, and both gene rearrangements in 46.7%. Among the AML cases, the IgH rearrangement occurred in 72.0%, the TCRγ rearrangement in 68.0%, and both gene rearrangements in 60.0%. In the lymphoma cases, the IgH rearrangement occurred in 93.1%, the TCRγ rearrangement in 51.7%, and both gene rearrangements in 44.8%. In the negative control group, the 10 cases were all negative. There was the phenomenon of "sequence-non-fidelity" in the hematologic malignancies; the detection rate of both genes was much higher than that of the single gene. The application of the RQ-PCR method in the detection of IgH and TCRγ gene rearrangements in hematologic malignancies has important clinical significance in MRD monitoring.

Epidemiological analysis of pneumoconiosis in the Xinjiang Uygur Autonomous Region and cases reported by the Urumqi Railway Bureau.

This study investigated the incidence and development of pneumoconiosis in the Xinjiang Uygur Autonomous Region and cases reported by the Urumqi Railway Bureau to provide a scientific basis for developing prevention and control measures against pneumoconiosis. Data from pneumoconiosis cases were input into Excel and analyzed by SPSS version 17.0. There were 13,165 cases of pneumoconiosis through 2010. Coal workers accounted for the largest proportion of cases. From July 2006 through 2010, a total of 1233 new cases of pneumoconiosis were reported in the Xinjiang Uygur Autonomous Region; most cases were reported in Urumqi. From 1981 to 2012, 3332 new cases of pneumoconiosis had been confirmed by the Urumqi Railway Bureau, including 77.73, 16.96, and 5.31% stage I, II, and III cases, respectively. In the last 30 years, the number of new pneumoconiosis cases peaked in 1986; most of them were silicosis cases. In addition, there were more than 200 cases of pneumoconiosis combined with pulmonary tuberculosis reported by the Urumqi Railway Bureau. The coal industry in Urumqi is the main industry in which occupational pneumoconiosis occurs in Xinjiang. Thus, substantial effort is still required to eliminate pneumoconiosis by 2030.

Bioinformatic analysis of phage AB3, a phiKMV-like virus infecting Acinetobacter baumannii.

The phages of Acinetobacter baumannii has drawn increasing attention because of the multi-drug resistance of A. baumanni. The aim of this study was to sequence Acinetobacter baumannii phage AB3 and conduct bioinformatic analysis to lay a foundation for genome remodeling and phage therapy. We isolated and sequenced A. baumannii phage AB3 and attempted to annotate and analyze its genome. The results showed that the genome is a double-stranded DNA with a total length of 31,185 base pairs (bp) and 97 open reading frames greater than 100 bp. The genome includes 28 predicted genes, of which 24 are homologous to phage AB1. The entire coding sequence is located on the negative strand, representing 90.8% of the total length. The G+C mol% was 39.18%, without areas of high G+C content over 200 bp in length. No GC island, tRNA gene, or repeated sequence was identified. Gene lengths were 120-3099 bp, with an average of 1011 bp. Six genes were found to be greater than 2000 bp in length. Genomic alignment and phylogenetic analysis of the RNA polymerase gene showed that similar to phage AB1, phage AB3 is a phiKMV-like virus in the T7 phage family.

Effects of acrylonitrile on lymphocyte lipid rafts and RAS/RAF/MAPK/ERK signaling pathways.

Acrylonitrile (ACN) is a widely used chemical in the production of plastics, resins, nitriles, acrylic fibers, and synthetic rubber. Previous epidemiological investigations and animal studies have confirmed that ACN affects the lymphocytes and spleen. However, the immune toxicity mechanism is unknown. Lipid rafts are cell membrane structures that are rich in cholesterol and involved in cell signal transduction. The B cell lymophoma-10 (Bcl10) protein is a joint protein that is important in lymphocyte development and signal pathways. This study was conducted to examine the in vitro effects of ACN. We separated lipid rafts, and analyzed Bcl10 protein and caveolin. Western blotting was used to detect mitogen-activated protein kinase (MAPK) and phosphorylated MAPK levels. The results indicated that with increasing ACN concentration, the total amount of Bcl10 remained stable, but was concentrated mainly in part 4 to part 11 in electrophoretic band district which is high density in gradient centrifugation. Caveolin-1 was evaluated as a lipid raft marker protein; caveolin-1 content and position were relatively unchanged. Western blotting showed that in a certain range, MAPK protein was secreted at a higher level. At some ACN exposure levels, MAPK protein secretion was significantly decreased compared to the control group (P < 0.05). These results indicate that ACN can cause immune toxicity by damaging lipid raft structures, causing Bcl10 protein and lipid raft separation and restraining Ras-Raf-MAPK-extracellular signal-regulated kinase signaling pathways.

Structural and phylogenetic analysis of Pto-type disease resistance gene candidates in Hevea brasiliensis.

The tomato Pto gene encodes a serine/threonine kinase (STK) whose molecular characterization has provided valuable insights into the disease resistance mechanism of tomato. Therefore, Pto is considered as a promising candidate for engineering broad-spectrum pathogen resistance in this crop. In this study, a pair of degenerate primers based on conserved subdomains of plant STKs similar to the tomato Pto protein was used to amplify similar sequences in a hevea cultivar (Hevea brasiliensis Muell. Arg). A fragment of ~550 bp was amplified, cloned, and sequenced. The sequence analysis of several clones revealed 12 distinct sequences highly similar to STKs. Based on their significant similarity with the tomato Pto protein (BLASTX E value<3e-53), seven sequences were classified as Pto resistance gene candidates (Pto-RGCs). Multiple sequence alignment of the hevea Pto-RGC products revealed that these sequences contain several conserved subdomains present in most STKs, as well as several conserved residues that are crucial for Pto function. Moreover, phylogenetic analysis showed that the hevea Pto-RGCs clustered with Pto, suggesting a common evolutionary origin with this resistance gene. The Pto-RGCs isolated in this study represent a valuable sequence resource that could assist in the development of disease resistance in hevea.

Novel method to establish molecular identity using inter-simple sequence repeat markers in cotton (Gossypium hirsutum) cultivars.

Plant molecular identity (ID) is used to describe molecular characteristics of plants, which should contain all of the necessary information. Using inter-simple sequence repeat (ISSR) primers, molecular ID can be described in a way that reflects the polymerase chain reaction (PCR) conditions, annealing temperature, and the bands obtained in PCR amplification. A new complete molecular ID system is described in this study, which can be easily used and expanded to include more information. Using three cotton cultivars, we analyzed the products of PCR with ISSR primers and discussed the strategy for establishing their molecular ID. Using the segmented naming method, we designate the simple names and the full name systems of these three cultivars.

A novel nonsense mutation in the sedlin gene (SEDL) causes severe spondyloepiphyseal dysplasia tarda in a five-generation Chinese pedigree.

Spondyloepiphyseal dysplasia tarda (SEDT) is an X-linked recessive osteochondrodysplasia characterized by disproportionately short stature and degenerative joint disease. The objective of this study was to describe a novel nonsense mutation in the sedlin gene (SEDL) causing severe SEDT in a large Chinese pedigree. The clinical features of all affected individuals and female carriers were presented. Four affected males of the family were diagnosed with SEDT according to their clinical and radiological features. Direct DNA sequencing of SEDL was performed. Reverse-transcription polymerase chain reaction (RT-PCR) experiments of total RNA from blood lymphocytes were performed to confirm the defect in SEDL. DNA sequencing revealed that all of the affected males carried a nonsense mutation (c.61G>T) in SEDL that has not been previously reported. The c.61G>T mutation resulted in a premature translation termination codon (GAG>TAG) at amino acid position 21 (p.E21*), and was predicted to initiate the degradation of mutant transcripts through the nonsense-mediated mRNA decay pathway. Two female carriers showed typical sequencing chromatograms of a heterozygote. Following genetic counseling, individual IV7 gave birth to a healthy baby. Therefore, identification of the novel nonsense mutation (c.61G>T) in the SEDT family enables carrier detection, genetic counseling, and prenatal diagnosis. The detailed genotype/phenotype descriptions contribute to the SEDL mutation spectrum. The continued identification of mutations in SEDT patients will greatly aid further elucidation of the role of the sedlin protein in normal bone growth.

Confirmation of a purple-leaved plum graft hybrid.

Fifty-seven scions from an adult purple-leaved plum tree were grafted onto the crown of a 6-year-old Yuhuang plum tree and compared to the control of a non-grafted tree. The floral buds of the purple-leaved plum were fully removed before blossoming to avoid sexual hybridization between the two species. The seeds of the Yuhuang plum were picked in July and sown in the spring after stratification. Three, eleven and eight variants with purplish red leaves were found among the seedlings that grew from the seeds picked in 1999, 2000, and 2001, respectively. The ratio of variant occurrence ranged from 2.3 to 15.8%. Our results confirmed the observation of a graft hybrid by Luther Burbank.

hOGG1 exon7 polymorphism and gastric cancer in case-control studies of Japanese Brazilians and non-Japanese Brazilians.

Polymorphism of hOGG1 may be capable of serving as a genetic marker for individual susceptibility to various cancers because of its role in the repair of oxyradical DNA damage. We examined the distribution of the hOGG1 Ser326Cys polymorphism and its presumed correlation with gastric cancer risk in two case-control studies of different ethnic groups in São Paulo, Brazil. Potentially eligible Japanese (JB) and non-Japanese Brazilian (NJB) case subjects were defined as patients with newly diagnosed malignant neoplasms of the stomach in 13 hospitals in São Paulo. Ninety-six JBs and 236 NJBs were adopted as subjects. Two controls were matched for each JB case, and one control for each NJB case. The subjects were interviewed using a questionnaire and their blood samples were collected. A significant difference in the distribution of this polymorphism between the two ethnic groups was observed (chi(2)=58.3, P<0.01). The mutant type (Ser/Cys or Cys/Cys) was predominant (approximately 65%) in the JBs, but was only present in approximately 40% of the NJBs. Logistic regression analysis showed no significant increased risk for either the Ser/Cys or Cys/Cys type in either group. The odds ratios of the Cys allele for gastric cancer were 1.01 (95% confidence interval (CI): 0.52-1.93) in the JBs and 0.85 (95% CI: 0.57-1.26) in the NJBs. In the NJBs, a significant increased risk of smoking was shown only in the Ser/Ser type, and no increased risk was shown in the genotypes with the Cys allele. However, no statistically significant interactions were observed with smoking or other possible confounding factors. No statistically significant difference in the distribution of the polymorphism was observed between the intestinal type and diffuse type of gastric cancer in either the JBs or the NJBs. The ethnic difference in hOGG1 Ser326Cys polymorphism was much greater than the case-control difference, and this polymorphism is unlikely to be associated with gastric cancer.

Cytochrome P450 2E1 polymorphism in gastric cancer in Brazil: case-control studies of Japanese Brazilians and non-Japanese Brazilians.

Cytochrome P450 2E1 (Cyp2E1) is involved in the metabolic oxidation of carcinogenic nitroso compounds, including N-nitrosoamines. There is an RsaI polymorphism in the transcriptional regulatory region of this gene, and in vitro evidence suggests that the variant type of this polymorphic site has higher transcriptional activity but less chlorzoxazone-metabolizing activity. Interindividual differences in the metabolic capacity of Cyp2E1 are assumed to be associated with cancer susceptibility, but the results of the previous studies on the relation between Cyp2E1 RsaI polymorphism and cancer susceptibility have been inconsistent. Two case-control studies of gastric cancer in Japanese Brazilians (96 cases, 192 controls) and Brazilians not of Japanese ancestry (non-Japanese Brazilians; 236 cases, 236 controls) in São Paulo were designed to clarify the role of the Cyp2E1 RsaI genotype in susceptibility to gastric cancer after considering multifactorial environmental influences. The subjects with variant RsaI genotypes amounted to 47% (28 of 59) and 48% (64 of 133), respectively, of the Japanese cases and controls, and 6% (11 of 187) and 10% (19 of 192), respectively, of the non-Japanese cases and controls. As expected, a difference in the distributions of the two groups was observed. The odds ratio of the RsaI variant genotype of Cyp2E1 was 0.46 (95% confidence interval, 0.21-1.04) in the non-Japanese Brazilian population and 0.98 (95% confidence interval, 0.50-1.90) in the Japanese Brazilian population after adjusting for sex, age, tobacco use, and meat consumption. Additional adjustment for potential confounding factors did not change the odds ratio substantially. No significant interactions were observed between the polymorphism and environmental factors. In regard to the histological type of gastric cancer, the variant genotype was significantly more prevalent than the common genotype in Japanese subjects with diffuse type gastric cancer. Our study suggests that the Cyp2E1 RsaI polymorphism is associated with a reduced risk of gastric cancer, although how the assumed increase in Cyp2E1 expression produced by this polymorphism is related to a reduced risk of cancer remains unclear. The observations in this study are consistent with the recent observations of esophageal cancer in endemic areas of China.