b Picorna-like viruses in the Picornavirales order are a large group of positive-strand RNA viruses that include numerous important pathogens for plants, insects, and humans. In these viruses, nonstructural protein 2C is one of the most conserved proteins and contains ATPase activity and putative RNA helicase activity. Here we expressed 2C protein of Ectropis obliqua picorna-like virus (EoV; genus Iflavirus, family Iflaviridae, order Picornavirales) in a eukaryotic expression system and determined that EoV 2C displays ATP-independent nucleic acid helix destabilizing and strand annealing acceleration activity in a concentration-dependent manner, indicating that this picornaviral 2C is more like an RNA chaperone than like the previously predicted RNA helicase. Our further characterization of EoV 2C revealed that divalent metal ions, such as Mg 2؉ and Zn 2؉ , inhibit 2C-mediated helix destabilization to different extents. Moreover, we determined that EoV 2C also contains ATPase activity like that of other picornaviral 2C proteins and further assessed the functional relevance between its RNA chaperone-like and ATPase activities using mutational analysis as well as their responses to Mg 2؉ . Our data show that, when one of the two 2C activities was dramatically inhibited or almost abolished, the other activity could remain intact, showing that the RNA chaperone-like and ATPase activities of EoV 2C can be functionally separated. This report reveals that a picorna-like virus 2C protein displays RNA helix destabilizing and strand annealing acceleration activity, which may be critical for picornaviral replication and pathogenesis, and should foster our understanding of picorna-like viruses and viral RNA chaperones.

For double-stranded RNA (dsRNA) viruses in the family Reoviridae, their inner capsids function as the machinery for viral RNA (vRNA) replication. Unlike other multishelled reoviruses, cypovirus has a single-layered capsid, thereby representing a simplified model for studying vRNA replication of reoviruses. VP5 is one of the three major cypovirus capsid proteins and functions as a clamp protein to stabilize cypovirus capsid. Here, we expressed VP5 from type 5 Helicoverpa armigera cypovirus (HaCPV-5) in a eukaryotic system and determined that this VP5 possesses RNA chaperone-like activity, which destabilizes RNA helices and accelerates strand annealing independent of ATP. Our further characterization of VP5 revealed that its helix-destabilizing activity is RNA specific, lacks directionality and could be inhibited by divalent ions, such as Mg2+, Mn2+, Ca2+ or Zn2+, to varying degrees. Furthermore, we found that HaCPV-5 VP5 facilitates the replication initiation of an alternative polymerase (i.e. reverse transcriptase) through a panhandle-structured RNA template, which mimics the 5′-3′ cyclization of cypoviral positive-stranded RNA. Given that the replication of negative-stranded vRNA on the positive-stranded vRNA template necessitates the dissociation of the 5′-3′ panhandle, the RNA chaperone activity of VP5 may play a direct role in the initiation of reoviral dsRNA synthesis.

Viral replication and capsid assembly in the viruses in the order Picornavirales requires polyprotein proteolytic processing by 3C or 3C-like (3CL) proteases. We identified and characterized the 3CL protease of Ectropis obliqua virus (EoV) of the newly established family Iflaviridae (order Picornavirales). The bacterially expressed EoV 3CL protease domain autocatalytically released itself from larger precursors by proteolytic cleavage, and cleavage sites were determined via N-terminal sequencing of the cleavage products. This protease also mediated trans-proteolytic activity and cleaved the polyprotein at the same specific positions. Moreover, we determined the critical catalytic residues (H2261, D2299, C2383) for the protease activity, and characterized the biochemical properties of EoV 3CL and its responses to various protease inhibitors. Our work is the first study to identify an iflaviral 3CL protease and further characterize it in detail and should foster our understanding of EoV and other iflaviruses.

Autophagy is a highly conserved process by which the cell contents are delivered to lysosomes for degradation, or are used to provide macromolecules for energy generation under conditions of nutritional starvation. It has previously been demonstrated that cancer cells in hypoxic regions, with an oxygen concentration below the normal physiological level, express hypoxia inducible factor (HIF)-1α, in order to adapt and survive. HIF-1α is important in the regulation of oxygen homeostasis and the transcription of hundreds of genes in response to conditions of hypoxia, hence maintaining energy and redox homeostasis. To determine if HIF-1α modulates autophagy and the underlying molecular mechanisms regulating this process, the human esophageal cancer EC109 and IMR90 human diploid fibroblast cell lines were exposed to normoxic or hypoxic conditions and the expression levels of various proteins subsequently examined. Small interfering RNA was used to silence p27, in order to investigate its role in the process of HIF-1α regulated autophagy. Hypoxia induced autophagy in IMR90 cells and it was revealed that immature IMR90 cells demonstrated an increased rate of autophagy compared with mature cells. HIF-1α promoted EC109 cell autophagy via positively modulating p27, whereas silencing of p27 abolished the autophagy induced by hypoxia. The present study identified the primary components of the p27-E2F1 signaling pathway by which HIF-1α regulates autophagy. A previously unidentified mechanism is here presented, via which cancer cells may generate energy, or obtain macromolecules for survival.

Background:Forkhead box P3 (Foxp3) plays important roles in the development and pathogensis of cancer. To investigate the association of 3 polymorphisms of Foxp3 (rs3761548, rs 3761549 and rs2280883) and cancer risk, an updated meta-analysis was performed.Methods:Around 11 studies including 4344 cancer patients and 4665 healthy controls were selected for this meta-analysis. There were nine studies with 3783 cases and 4096 controls for rs3761548, 4 studies with 1669 cases and 1613 controls for rs3761549 and 4 studies with 1821 cases and 1799 controls for rs2280883. Odds radios (ORs) and 95% confidence intervals (CIs) were used to evaluate the cancer risk.Results:Meta-analysis showed that rs3761548 was associated with an increased cancer risk in the overall population under the recessive model (AA vs CA + CC: OR = 1.45, 95%CI = 1.03–2.02, P = .03). No association was found between rs3761549, rs2280883 polymorphisms, and cancer susceptibility in the overall population. Nonetheless, in the genotyping methods subgroup analysis of rs2280883, a lower risk of cancer was found in studies using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) under the allelic model (C vs T: OR = 0.70, 95%CI = 0.52–0.95, P = .02), heterozygote model (TC vs TT: OR = 0.60, 95%CI = 0.41–0.87, P = .008) and dominant model (CC + TC vs TT: OR = 0.63, 95%CI = 0.45–0.90, P = .01). In the subgroup analysis by cancer types showed C allele or TC carriers were insusceptible to cancer under 3 genetic models (C vs T: OR = 0.78, 95%CI = 0.64–0.95, P = .01; TC vs TT: OR = 0.50, 95%CI = 0.32–0.79, P = .003; CC + TC vs TT: OR = 0.64, 95%CI = 0.51–0.82, P < .001).Conclusion:Our results suggest that rs3761548 polymorphism is associated with cancer risk.

ABSTRACT. Iron metabolism plays an important role in the pathogenesis of lung cancer. This study aimed to investigate the effect of gene silencing of iron regulatory protein-2 (IRP2) on mRNA and protein expression of transferrin (Tf), transferrin receptor (TfR), and ferritin (Fn) in A549 lung cancer cells. A549 cells were cultured and divided into a liposome control group, a liposome + oligonucleotide (SCODN) control group, and a Lipofectamine + antisense oligonucleotide (ASODN) group. RT-PCR and Western blotting were used to detect mRNA and protein expression of Tf, TfR, and Fn. We found no significant change in Tf mRNA expression among the 3 groups (P = 0.078). TfR and Fn mRNA expressions in the ASODN group notably decreased compared to the liposome and SCODN groups (P < 0.01). IRP2 and TfR protein expressions in the ASODN group were significantly lower than in the liposome or SCODN groups (P < 0.05), whereas no significant change in Tf protein expression was observed between the 3 groups (P = 0.088). Fn protein expression in the ASODN group was significantly higher 5515 ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 13 (3): 5514-5522 (2014) Regulation of iron metabolism by IRP2 than in the liposome or SCODN group (P < 0.05). IRP2 can regulate the expression of TfR and Fn by changing its own protein expression and thereby regulate iron metabolism.

ABSTRACT. The aim of this study was to investigate the association between four single nucleotide polymorphisms in NR3C1 (Tth111I, BclI, ER22/23EK, and N363S), which encode the glucocorticoid receptor, and asthma susceptibility in patients from the Henan Province of China. Three hundred and twenty-eight patients with asthma and 60 healthy volunteers were recruited to this study. The target SNPs were genotyped by polymerase chain reaction (PCR)-high resolution melting and PCR-restriction fragment length polymorphism. The frequencies of the AA (8.84%) and GG (30.79%) genotypes of Tth111I were higher, and that of the AG genotype was lower (60.37%), in the asthma patients compared to that seen in healthy controls (5.00, 26.67, and 68.33%, respectively). On the other hand, asthma patients showed higher frequencies of the AA genotype (78.05%) of N363S, and lower frequencies of the AG and GG genotypes (15.55 and 6.40%), compared to healthy volunteers (71.67, 18.33, and 10.00%, respectively). Neither of these differences were found to be statistically significant. Moreover, we observed no significant differences in the genotype or allele frequencies of the BclI and ER22/23EK SNPs between the patient and control groups. In conclusion, SNPs in NR3C1 were not significantly associated with asthma in patients from the Henan Province. Patients showed higher frequencies of the AA and GG genotypes of Tth111I and the AA genotype of the N363S SNP compared to healthy volunteers, although these differences were not significant.

Background: LncRNA metastasis associated with lung adenocarcinoma transcript-1 (MALAT1) was involved in pathogenesis and progress of diverse cancers. To investigate the association of MALAT1 and cancer susceptibility, this meta-analysis was appraised.Methods: 12 studies including 7007 cancer patients and 8791 controls were selected for this meta-analysis. Ratio radiation (ORS) and 95% confidence interval (CIS) were used to assess cancer susceptibility.Results: There was no significant association between rs3200401 polymorphism and the risk of cancer. However, rs3200401 was correlated with an increased risk of digestive cancer in allelic model (OR=1.15, 95%CI=1.04-1.28, P=0.009) and dominant model (OR=1.16, 95%CI=1.02-1.31, P=0.02). There was a borderline association between rs664589 and cancer susceptibility under the dominant model (OR=1.17, 95%CI=1.00-1.38, P=0.05). Rs619586 was associated with decreased cancer risk in all populations under four models (G vs A: OR=0.86, 95%CI=0.78-0.94, P=0.001; GG vs AA: OR=0.60, 95%CI=0.42-0.84, P=0.003; GG+AG vs AA: OR=0.87, 95%CI=0.78-0.97, P=0.009; GG vs AG+AA: OR=0.61, 95%CI=0.44-0.84, P=0.003). Moreover, rs1194338 was decreased associated with cancer susceptibility (A vs C: OR=0.89, 95%CI=0.80-0.98, P=0.01; AA vs CC: OR=0.77, 95%CI=0.62-0.96, P=0.02; AA+AC vs CC: OR=0.87, 95%CI=0.77-1.00, P=0.04; AA vs AC+CC: OR=0.82, 95%CI=0.67-1.00, P=0.05).Conclusion: Our results suggest that rs619586 and rs1194338 are associated with decreased cancer risk, while rs3200401 and rs664589 correlated with increased digestive cancer risk.