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Background: Long non-coding RNA growth arrest-specific transcript 5 (lncRNA GAS5) is a well-known tumor suppressor in the pathogenesis of a variety of human cancers. The precise role of GAS5 in pancreatic cancer (PC) progression is currently unknown, so the aim of this study was to explore the functional participation of GAS5 in PC metastasis., Methods: The expression changes of GAS5, miR-32-5p and PTEN in human PC specimens and cell lines were compared by means of molecular biology methods. Transfection of the recombinant plasmid was applied to modulate the expression levels of the target genes. RIP and RNA pull-down assays were designed to investigate the interaction between GAS5 and miR-32-5p. The effect of GAS5 and miR-32-5p on PC progression was assessed with cell proliferation, migration, invasion and apoptosis in vitro., Results: GAS5 and PTEN protein were decreased in human PC tissues and cells, but miR-32-5p was increased. GAS5 induction greatly inhibited the proliferation, migration and invasion of PC cells PANC-1 and BxPC-3 in vitro and simultaneously induced cell apoptosis. Moreover, GAS5 positively regulated the expression of PTEN through miR-32-5p. Furthermore, GAS5 suppressed the proliferation, migration and invasion of PC cells through regulating miR-32-5p/PTEN axis. Additionally, this finding was further supported by the results of in vivo experiments., Conclusion: GAS5 could positively regulate PTEN-induced tumor-suppressor pathway via miR-32-5p, thereby suppressing PC metastasis.

Background: Sorghum is mainly used as a human food and beverage source, playing an important role in the production of ethanol and other bio-industrial products. Thus it is regarded as a model crop for energy plants. Genetic map construction is the foundation for marker-assisted selection and gene cloning. So far several sorghum linkage maps have been reported using different kinds of molecular markers. However marker numbers and chromosome coverage are limited. As a result, it is difficult to get consistent results and the maps are hard to unify. In the present study, the genomes of 130 individuals consisting an F 2 population together with their parents were surveyed using a high-throughput sequencing technique. A high-density linkage map was constructed using specific-locus amplified fragments (SLAF) markers. This map can provide information and serve as a reference for effective gene exploration, and for marker assisted-breeding program., Results: A high-throughput sequencing method was adopted to screen SLAF markers with 130 F 2 individuals from a cross between a grain sorghum variety, J204, and a sweet sorghum variety, Keter. In the present study, 52,928 suitable SLAF markers out of 43,528,021 pair-end reads were chosen to conduct genetic map construction, 12.0% of which were polymorphic. Among the 6353 polymorphic SLAF markers, 5829 (91.8%) were successfully genotyped in the F 2 mapping population. Finally 2246 SLAF markers were obtained to construct a high-density genetic linkage map. The total distance of linkage map covering all 10 chromosomes was 2158.1 cM. The largest gap on each chromosome was 10.2 cM on average. The proportion of gaps less than and/or equal to 5.0 cM was averagely 98.1%. The markers on each chromosome ranged from 123 (chromosome 9) to 315 (chromosome 4) with a mean value of 224.6, the distance between adjacent markers ranged from 0.6 (chromosome 10) to 1.3 cM (chromosome 9) with an average distance of only 0.98 cM., Conclusion: A high density sorghum genetic map was constructed in this study. The total length was 2158.1 cM covering all 10 chromosomes with a total number of 2246 SLAF markers. The construction of this map can provide detailed information for accurate gene localization and cloning and application of marker-assisted breeding.

Background: Trichinellosis is a re-emerging infectious disease, caused by Trichinella spp. Cathepsin F belongs to cysteine protease that is a major virulence factor for parasitic helminths, and it may be a potential anti-helminth drug target and vaccine candidate. The aim of this study was to clone, express and identify a cathepsin F-like protease in Trichinella spiralis and to investigate its biochemical characteristics., Methods: The full-length cDNA encoding a putative cathepsin F-like protease in T. spiralis, TsCF1, was cloned and its biochemical characterization and expression profile were analyzed. Transcription of TsCF1 at different developmental stages of T. spiralis was observed by RT-PCR. The recombinant TsCF1 protein was expressed by prokaryotic expression system and recombinant TsCF1 (rTsCF1) was analyzed by western blotting. And expression of TsCF1 at muscle larvae stage was performed by immunofluorescent technique. Molecular modeling of TsCF1 and its binding mode with E-64 and K11777 were analyzed. Enzyme activity and inhibitory test with E-64 as inhibitor were investigated by using Z-Phe-Arg-AMC as specific substrate., Results: Sequence analysis revealed that TsCF1 ORF encodes a protein of 366 aa with a theoretical molecular weight of 41.9 kDa and an isoelectric point of 7.46. The cysteine protease conserved active site of Cys173, His309 and Asn333 were identified and cathepsin F specific motif ERFNAQ like KLFNAQ sequence was revealed in the propeptide of TsCF1. Sequence alignment analysis revealed a higher than 40 % identity with other cathepsin F from parasitic helminth and phylogenetic analysis indicated TsCF1 located at the junction of nematode and trematode. RT-PCR revealed the gene was expressed in muscle larvae, newborn larvae and adult stages. SDS-PAGE revealed the recombinant protein was expressed with the molecular weight of 45 kDa. The purified rTsCF1 was used to immunize rabbit and the immune serum could recognize a band of about 46 kDa in soluble protein of adult, muscle larvae and ES product of muscle larvae. Immunolocalization analysis showed that TsCF1 located on the cuticle and stichosome of the muscle larvae. After renaturation rTsCF1 demonstrated substantial enzyme activity to Z-Phe-Arg-AMC substrate with the optimal pH 5.5 and this activity could be inhibited by cysteine protease inhibitor E-64. Further analysis showed the kinetic parameters of rTsCF1 to be Km = 0.5091 μM and Vmax = 6.12 RFU/s μM at pH 5.5, and the IC50 value of E64 was 135.50 ± 16.90 nM., Conclusion: TsCF1 was expressed in all stages of T. spiralis and localized in the cuticle and stichosome. TsCF1 might play a role in the life cycle of T. spiralis and could be used as a potential vaccine candidate and drug target against T. spiralis infection.

Background: Gossypium raimondii is a Verticillium wilt-resistant cotton species whose genome encodes numerous disease resistance genes that play important roles in the defence against pathogens. However, the characteristics of resistance gene analogues (RGAs) and Verticillium dahliae response loci (VdRLs) have not been investigated on a global scale. In this study, the characteristics of RGA genes were systematically analysed using bioinformatics-driven methods. Moreover, the potential VdRLs involved in the defence response to Verticillium wilt were identified by RNA-seq and correlations with known resistance QTLs., Results: The G. raimondii genome encodes 1004 RGA genes, and most of these genes cluster in homology groups based on high levels of similarity. Interestingly, nearly half of the RGA genes occurred in 26 RGA-gene-rich clusters (Rgrcs). The homology analysis showed that sequence exchanges and tandem duplications frequently occurred within Rgrcs, and segmental duplications took place among the different Rgrcs. An RNA-seq analysis showed that the RGA genes play roles in cotton defence responses, forming 26 VdRLs inside in the Rgrcs after being inoculated with V. dahliae. A correlation analysis found that 12 VdRLs were adjacent to the known Verticillium wilt resistance QTLs, and that 5 were rich in NB-ARC domain-containing disease resistance genes., Conclusions: The cotton genome contains numerous RGA genes, and nearly half of them are located in clusters, which evolved by sequence exchanges, tandem duplications and segmental duplications. In the Rgrcs, 26 loci were induced by the V. dahliae inoculation, and 12 are in the vicinity of known Verticillium wilt resistance QTLs.

Background: To evaluate the role of intensity modulated radiotherapy (IMRT) for locally advanced pancreatic cancer (LAPC) and metastatic pancreatic cancer (MPC), and the prognostic factors in the setting of multidisciplinary approach strategies., Methods: 63 patients with LAPC and MPC receiving IMRT in our institution were retrospectively identified. Information on patient baseline, treatment characteristics and overall survival (OS) time were collected. Data of pain relief and toxicity were evaluated. Univariate and multivariate analyses were conducted to investigate the prognostic factors., Results: All patients received IMRT with a median dose of 46.0 Gy. The median OS for LAPC and MPC patients were 15.7 months and 8.0 months, respectively (p = 0.029). Symptomatic improvements were observed in the 44 patients with abdominal/back pain after radiotherapy (RT) or concurrent chemoradiotherapy (CCRT), particularly in those with severe pain. Only 13.9% and 14.8% cases presented Grade ≥ 3 hematologic toxicities in RT and CCRT group, while no cases developed Grade ≥ 3 non-hematologic toxicities in both groups. Multivariate analysis indicated that tumors located in pancreas body/tail (HR 0.28, p = 0.008), pretreatment CA19-9 < 1000 U/mL (HR 0.36, p = 0.029) and concurrent chemotherapy (HR 0.37, p = 0.016) were independent favorable predictors for OS., Conclusions: CCRT further improved OS for LAPC and MPC with acceptable toxicities, and use of RT markedly alleviated pain. Tumors located in pancreas body/tail, pretreatment CA19-9 level of < 1000 U/mL and CCRT were associated with better OS. However, regional intra-arterial chemotherapy did not show any survival benefit in our study.

Background: Lung cancer is the leading cause of cancer-related death in the world, with metastasis as the main reason for the mortality. CELF1 is an RNA-binding protein controlling the post-transcriptional regulation of genes related to cell survival. As yet, there is little knowledge of CELF1 expression and biological function in lung cancer. This study investigated the expression levels of CELF1 in lung cancer tissues and the biological function of CELF1 in lung cancer cells., Methods: CELF1 mRNA expression was determined in lung cancer and normal tissues, and the relationship between the expression level of CELF1 and clinicopathological parameters was evaluated. The biological function of CELF1 in A549 and H1299 lung cancer cell lines growth was examined., Results: The expression of CELF1 was higher in human lung cancer tissues compared with the normal lung tissue. Lentiviral-mediated transfection of CELF1 siRNA effectively silenced the expression of CELF1 in both A549 and H1299 cells. Moreover, CELF1 knockdown markedly reduced the survival rate of lung cancer cells. Colony formation assays revealed a reduction in the number and size of lung cancer cell colonies from CELF1 knockdown., Conclusion: These results indicated that CELF1 may have significant roles in the progression of lung cancer, and suggested that siRNA mediated silencing of CELF1 could be an effective tool in lung cancer treatment.

Background: Existing standard non-molecular diagnostic methods such as viral culture and immunofluorescent (DFA) are time-consuming, labor intensive or limited sensitivity. Several multiplex molecular assays are costly. Therefore, there is a need for the development of a rapid and sensitive diagnosis of respiratory viral pathogens., Methods: A GeXP-based multiplex RT-PCR assay (GeXP assay) was developed to detect simultaneously sixteen different respiratory virus types/subtypes. Seventeen sets of chimeric primers were used to initiate the RT-PCR, and one pair of universal primers was used for the subsequent cycles of the RT-PCR. The specificity of the GeXP assay was examined with positive controls for each virus type/subtype. The sensitivity was evaluated by performing the assay on serial ten-fold dilutions of in vitro-transcribed RNA of all RNA viruses and the plasmids containing the Adv and HBoV target sequence. GeXP assay was further evaluated using 126 clinical specimens and compared with Luminex xTAG RVP Fast assay., Results: The GeXP assay achieved a sensitivity of 20-200 copies for a single virus and 1000 copies when all of the 16 pre-mixed viral targets were present. Analyses of 126 clinical specimens using the GeXP assay demonstrated that GeXP assay and the RVP Fast assay were in complete agreement for 109/126 (88.51%) of the specimens. GeXP assay was more sensitive than the RVP Fast assay for the detection of HRV and PIV3, and slightly less sensitive for the detection of HMPV, Adv, RSVB and HBoV. The whole process of the GeXP assay for the detection of 12 samples was completed within 2.5 hours., Conclusions: In conclusion, the GeXP assay is a rapid, cost-effective, sensitive, specific and high throughput method for the detection of respiratory virus infections.

Background: EV71 is associated with the fatal cases of brain stem encephalitis during large HFMD outbreaks from 1998 to 2008. EV71 may continuously shed from upper respiratory tracts and feces of HFMD patients for relatively long time after recovery. However, the persistence of viruses in the patients' secretions and excretions is not clear., Methods: Serial throat swabs and feces of 34 definitely diagnosed patients, including 30 mild cases and 4 severe cases, were traced and collected with the interval of 2 to 4 days for up to 32 and 48 days, respectively, and tested by a nested RT-PCR., Results: The EV-71 specific sequences were identified by a Nested RT-PCR in all specimens of 0-4 days, and 5-8 days. The positive rates of EV71 in throat swabs dropped markedly to 42.86% during 9-12 days, and maintained at 20-30% during 13-24 days, while that in feces reduced to 71.43% during 9-12 days, and maintained roughly 20% till 37-40 days. EV71 nucleotide of 36.36% cases disappeared simultaneously both in throats and feces, 39.39% cases showed longer persistence of EV71 nucleotides in feces, and 21.21% were longer in throats. The longest duration of shedding observed was 24 days for throat swabs and 42 days for fecal specimens., Conclusions: EV71 shedding from respiratory tract may continue for nearly four weeks after onset, but its excretion through feces can persist more than five weeks.

Background: The rapid and accurate detection and identification of the new subtype of the pathogens is crucial for diagnosis, treatment and control of the contagious disease outbreak. Here, in this study, an approach to detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139 was established using oligonucleotide microarray. We coupled multiplex PCR with oligonucleotide microarray to construct an assay suitable for simultaneous identification of two subtypes of the pathogens., Results: The stx1, stx2 gene and uidA gene having the specific mutant spot were chosen as the targets for Escherichia coli O157:H7, and meanwhile the ctxA, tcpA, and LPSgt gene for Vibrio cholerae O139. The oligonucleotide microarray was composed of eight probes including negative control and positive control from 16S rDNA gene. The six primers were designed to amplify target fragments in two triplex PCR, and then hybridized with oligonucleotide microarray. An internal control would be to run a PCR reaction in parallel. Multiplex PCR did not produce any non-specific amplicons when 149 related species or genera of standard bacteria were tested (100% specificity). In addition, Escherichia coli O157:H7 and Escherichia coli O157:non-H7, Vibrio cholerae O139 and Vibrio cholerae O1 had been discriminated respectively. Using recombinant plasmid and target pathogens, we were able to detect positive hybridization signals with 102 copies/muL and 103 cfu/mL per reaction., Conclusion: The DNA microarray assay reported here could detect and identify Escherichia coli O157:H7 and Vibrio cholerae O139, and furthermore the subtype was distinguished. This assay was a specific and sensitive tool for simultaneous detection and identification of the new subtype of two pathogens causing diarrhea in human.

Background: RNA-specific adenosine deaminase ADAR1 is ubiquitously expressed in a variety of mammalian cells and tissues. Although its physiological importance in non-nervous tissues has been confirmed by analysis of null mutation phenotypes, few endogenous editing substrates have been identified in numerous peripheral tissues and biological function of ADAR1 has not been fully understood., Methods: A conditional site-specific, ribozyme-based gene knock-down strategy was utilized to study the function of full-length isoform of ADAR1 (p150 protein) in HeLa cell. Double-stable HeLa cell lines were developed by transfecting HeLa Tet-On cells with a pTRE-derived plasmid that can express a hammerhead ribozyme against mRNA of p150 ADAR1 isoform under induction condition. Semi-quantitative RT-PCR and Western blotting were performed to measure the expression of p150 in selected cell clones. Cell proliferation was evaluated by means of MTT assay and growth curve analysis. Cellular morphological changes were observed under light microscope. Flow Cytometry was used for cell cycle analysis. Growth rate of cell transplants in BALB/c nude mice was also investigated., Results: Both HeLa cell proliferation in vitro and the growth rate of transplanted HeLa cell-derived tumors in nude mice in vivo were significantly inhibited due to reduced expression of ADAR1 p150. Additionally, cell cycle analysis showed that cell progression from G1 phase to S phase was retarded in the ADAR1 p150 suppressed cells., Conclusion: Our results suggest that normal expression and functioning of p150 ADAR1 is essential for the maintenance of proper cell growth. The mechanisms underlying ADAR1's action might include both editing of currently unknown double-stranded RNAs and interacting with other cellular dsRNA-related processes.