Intron losses and gains in the nematodes.

BACKGROUND: The evolution of spliceosomal introns has been widely studied among various eukaryotic groups. Researchers nearly reached the consensuses on the pattern and the mechanisms of intron losses and gains across eukaryotes. However, according to previous studies that analyzed a few genes or genomes, Nematoda seems to be an eccentric group. RESULTS: Taking advantage of the recent accumulation of sequenced genomes, we extensively analyzed the intron losses and gains using 104 nematode genomes across all the five Clades of the phylum. Nematodes have a wide range of intron density, from less than one to more than nine per kbp coding sequence. The rates of intron losses and gains exhibit significant heterogeneity both across different nematode lineages and across different evolutionary stages of the same lineage. The frequency of intron losses far exceeds that of intron gains. Five pieces of evidence supporting the model of cDNA-mediated intron loss have been observed in ten Caenorhabditis species, the dominance of the precise intron losses, frequent loss of adjacent introns, high-level expression of the intron-lost genes, preferential losses of short introns, and the preferential losses of introns close to 3'-ends of genes. Like studies in most eukaryotic groups, we cannot find the source sequences for the limited number of intron gains detected in the Caenorhabditis genomes. CONCLUSIONS: These results indicate that nematodes are a typical eukaryotic group rather than an outlier in intron evolution.

Transplantation of a Novel Recombinant Adeno-Associated Virus (pAAV-HE1B19K-TE1A) Demonstrates Higher Anti-Tumor Effects in Tumor Cells.

BACKGROUND Oncolytic viruses (OVs) can specifically infect and kill tumor cells. Adeno-associated virus (AAV) is a widely-studied OV. This study aimed to construct a tumor-targeted recombinant AAV using genetic engineering technology. MATERIAL AND METHODS The transgene plasmid pAAV-HE1B19K-TE1A was constructed with 4 genes (hTERT, E1A, HKII, and E1B19K) and co-transfected with pAAV-RC and pHelper to tumor cells (HepG2, A549, BGC-803) and normal cells (HUVEC). rAAV was verified with fluorescence microscopy. Quantitative PCR (qPCR) assay was used to test the titer of rAAV in each cell line. Apoptosis was analyzed using qPCR and Western blot assay. MTT was used to detect the effect of rAAV on cell viability. RESULTS The pAAV-HE1B19K-TE1A transgene plasmid was successfully structured. pAAV-HE1B19K-TE1A was highly expressed in all tumor cells. The titers of pAAV-HE1B19K-TE1A in HepG2, A549, and BGC-803 were 7.4×107, 1.4×108, and 1.1×108 gc/µl, respectively. pAAV-HE1B19K-TE1A significantly decreased cell viability of tumor cells compared to that in HUVEC (p<0.05). pAAV-HE1B19K-TE1A remarkably triggered cleaved caspase 3 (C-caspase 3) activity in tumor cells compared to that in untransfected tumor cells (p<0.05). pAAV-HE1B19K-TE1A significantly induced release of cytochrome C (Cyto C) in tumor cells compared to that in untransfected tumor cells (p<0.05). pAAV-HE1B19K-TE1A demonstrated no toxicity to vital tissues of animals. CONCLUSIONS Tumor-targeted rAAV was successfully produced using the Helper-free system with recombinant plasmid, demonstrating high efficacy in decreasing viability of tumor cells without adverse effects on normal cells.

Using the entrapment sequence method as a standard to evaluate key steps of proteomics data analysis process.

BACKGROUND: The mass spectrometry based technical pipeline has provided a high-throughput, high-sensitivity and high-resolution platform for post-genomic biology. Varied models and algorithms are implemented by different tools to improve proteomics data analysis. The target-decoy searching strategy has become the most popular strategy to control false identification in peptide and protein identifications. While this strategy can estimate the false discovery rate (FDR) within a dataset, it cannot directly evaluate the false positive matches in target identifications. RESULTS: As a supplement to target-decoy strategy, the entrapment sequence method was introduced to assess the key steps of mass spectrometry data analysis process, database search engines and quality control methods. Using the entrapment sequences as the standard, we evaluated five database search engines for both the origanal scores and reprocessed scores, as well as four quality control methods in term of quantity and quality aspects. Our results showed that the latest developed search engine MS-GF+ and percolator-embeded quality control method PepDistiller performed best in all tools respectively. Combined with efficient quality control methods, the search engines can improve the low sensitivity of their original scores. Moreover, based on the entrapment sequence method, we proved that filtering the identifications separately could increase the number of identified peptides while improving the confidence level. CONCLUSION: In this study, we have proved that the entrapment sequence method could be an useful strategy to assess the key steps of the mass spectrometry data analysis process. Its applications can be extended to all steps of the common workflow, such as the protein assembling methods and data integration methods.

HGF protects cultured cortical neurons against hypoxia/reoxygenation induced cell injury via ERK1/2 and PI-3K/Akt pathways.

Hepatocyte growth factor (HGF) has been revealed to exert multipotent activities on a variety of cells. In this study, we investigated whether HGF had a direct neuroprotection on cultured cerebral cortical neurons subjected to hypoxia/reoxygenation (H/R) and explored the intracellular signalings mediated the effects. The decrease in cell viability and increase in number of apoptotic cells resulting from H/R were significantly prevented by HGF pre-treatment. HGF stimulated both ERK1/2 and Akt activities in cortical neurons. Inhibition of ERK activation completely abolished the protective effects of HGF, and inhibition of Akt activation reduced, but did not completely eliminate the HGF mediated neuroprotection. It is suggested that the neuroprotection of HGF depend on ERK1/2 pathway, and, to a lesser extent, PI-3K/Akt pathway. In addition, we found that pre-treatment with HGF remarkably attenuated the decrease in expression of Bcl-2 and Bcl-xL induced by H/R, but failed to affect the amount of Bax. It is likely that Bcl-2 and Bcl-xL contribute to the protective effects of HGF.

The p53 network: p53 and its downstream genes.

The tumor-suppressor gene p53 and its downstream genes consist of a complicated gene network. p53 is a key molecular node in the network, which is activated in response to several cellular signals resulting in the maintenance of genetic stability. Several cellular signals may activate the p53 network. When the expression of P53 is elevated, P53-MDM2 module and the ubiquitin system can accurately regulate the expression level of P53. P53 can bind to specific DNA sequence, activate its downstream genes expression, and control cell-cycle arrest, DNA repair, and apoptosis. Elucidating the function of p53 gene network will help understand the interaction mechanisms of p53 and its downstream genes.

[Effect of polysaccharides content of tissue culturing seedlings on Dendrobium candidium under sound wave stimulation].

OBJECTIVE: To detect the polysaccharides content of tissue culturing seedlings on Dendrobium candidium under special sound wave stimulation. METHODS: The content of polysaccharides was detected by phenol-vitriol colorimetry method. RESULTS: The polysaccharides content of the groups stimulated continuously for 24d was higher obviously than the content of the control groups. CONCLUSION: The stimulation of the special sound wave promoted markedly the synthesis of polysaccharides in D. candidium. It may affect obviously the metabolic pathway of polysaccharides in D. candidium.

[A comparative study on isoenzymes of Sarcocystis spp. from cattle and water buffaloes].

OBJECTIVE: To analyze the zymogram of peroxidase (PER) and phosphoglucose isomerase (PGI) of three species of Sarcocystis. METHODS: The collected parasites were homogenized and fragmented by ultrasonication. After centrifugation, the supernatants were analyzed by isoelectric focusing electrophoresis. RESULTS: The isolates of S. cruzi from infected water buffalo and cattle all showed identical enzyme profiles, 7 bands of PER at pH 4.44-6.98 and 6 bands of PGI at pH 4.66-6.53; and same with the isolates of S. hirsuta. 5 bands of PER at pH 4.97-7.15 and 4 bands of PGI at pH 4.70-6.51. The zymograms among S. cruzi, S. hirsuta and S. fusiformis were different considerably. CONCLUSION: The data support the hypothesis that both water buffalo and cattle are the natural intermediate hosts of S. cruzi and S. hirsuta at the gene level. S. cruzi, S. hirsuta and S. fusiformis are different species.

Characterization of the human PAP1 gene and its homologue possible involvement in mouse embryonic development.

We have identified PAP1 gene, a novel member of the immunoglobulin superfamily (IGSF) from U251-pTet-p53 cell line, which carried a wild-type p53 transgene. The gene has been localised to chromosome 16p12-13. Alignment of the predicted protein sequence for Human, Pan troglodytes, Canis, Mus musculus and Gallus gallus revealed it was highly conserved. Its homologue, IGSF6, possible involves in mouse embryonic development. The presence of IGSF6 specific transcript was detected by Northern blot in the RNAs extracted from 11 to 14 day postconception. IGSF6 expression is different in mouse embryos of the different ages. In situ hybridization performed on mice embryos sections showed the differential presence of IGSF6 in developing lung and kidney. This structure and differential expression suggests a function involvement in embryonic development, perhaps involvement in cell proliferation.

Effects of exogenous p53 transfection on the gene expression in the human brain glioma cell line U251.

The p53 gene is activated in response to several malignancy-associated stress signals by transactivation of downstream genes and by transcription-independent mechanisms. In order to identify new p53 downstream genes, we established a new system of p53 gene inducible expression, U251-pTet-p53 cell line, with the Tet-On Gene Expression System, in which exogenous p53 gene could overexpress in doxycycline (Dox) medium but not in the medium without Dox. By comparing their random primer RT-PCR products, it was proved that exogenous p53 gene expression could lead to many genes differential expression, some up-expressed and others down-expressed. All of these differential expressed genes may be p53 downstream genes. We can gain the magnitude of p53 downstream genes, which provides the basis of directly cloning of novel p53 downstream genes and further studying of p53 regulatory network.