Detection of picobirnaviruses in vervet monkeys (Chlorocebus sabaeus): Molecular characterization of complete genomic segment-2.

During 2014-2015, 270 fecal samples were collected from non-diarrheic, captive and wild African green monkeys (AGMs) on the island of St. Kitts, Caribbean region. By RNA-PAGE, picobirnaviruses (PBVs) were detected in sixteen captive AGMs. By RT-PCR and sequencing of partial gene segment-2, PBVs in 15 of these 16 samples were assigned to genogroup-I. The full-length nucleotide (nt) sequence of gene segment-2 of one of the genogroup-I PBV strains, strain PBV/African green monkey/KNA/016593/2015, was obtained using a non-specific primer-based amplification method with modifications. Gene segment-2 of strain 016593 was 1707bp long, and encoded a putative RNA-dependent RNA polymerase (RdRp) of 538aa. Furthermore, the nearly complete gene segment-2 sequences of three other AGM PBV strains were determined using primers designed from gene segment-2 sequence of 016593. The gene segment-2 of the 4 AGM PBV strains were almost identical to each other, and exhibited a high degree of genetic diversity (maximum nt and deduced aa sequence identities of 66.4% and 65.3%, respectively) with those of PBVs from other host species. The 5'- and 3'- (except for one mismatch) end nt sequences and the three domains of RdRps were retained in the AGM PBV strains. To our knowledge, this is the first report on detection, and molecular characterization of complete gene segment-2 of PBVs in vervet monkeys. PBVs were detected for the first time from the Caribbean region.

Role of the crystalline form of titanium dioxide nanoparticles: Rutile, and not anatase, induces toxic effects in Balb/3T3 mouse fibroblasts.

The wide use of titanium dioxide nanoparticles (TiO2 NPs) in industrial applications requires the investigation of their effects on human health. In this context, we investigated the effects of nanosized and bulk titania in two different crystalline forms (anatase and rutile) in vitro. By colony forming efficiency assay, a dose-dependent reduction of the clonogenic activity of Balb/3T3 mouse fibroblasts was detected in the presence of rutile, but not in the case of anatase NPs. Similarly, the cell transformation assay and the micronucleus test showed that rutile TiO2 NPs were able to induce type-III foci formation in Balb/3T3 cells and appeared to be slightly genotoxic, whereas anatase TiO2 NPs did not induce any significant neoplastic or genotoxic effect. Additionally, we investigated the interaction of TiO2 NPs with Balb/3T3 cells and quantified the in vitro uptake of titania using mass spectrometry. Results showed that the internalization was independent of the crystalline form of TiO2 NPs but size-dependent, as nano-titania were taken up more than their respective bulk materials. In conclusion, we demonstrated that the cytotoxic, neoplastic and genotoxic effects triggered in Balb/3T3 cells by TiO2 NPs depend on the crystalline form of the nanomaterial, whereas the internalization is regulated by the particle size.

Changes in Caco-2 cells transcriptome profiles upon exposure to gold nanoparticles.

Higher efficacy and safety of nano gold therapeutics require examination of cellular responses to gold nanoparticles (AuNPs). In this work we compared cellular uptake, cytotoxicity and RNA expression patterns induced in Caco-2 cells exposed to AuNP (5 and 30nm). Cellular internalization was dose and time-dependent for both AuNPs. The toxicity was observed by colony forming efficiency (CFE) and not by Trypan blue assay, and exclusively for 5nm AuNPs, starting at the concentration of 200µM (24 and 72h of exposure). The most pronounced changes in gene expression (Agilent microarrays) were detected at 72h (300µM) of exposure to AuNPs (5nm). The biological processes affected by smaller AuNPs were: RNA/zinc ion/transition metal ion binding (decreased), cadmium/copper ion binding and glutathione metabolism (increased). Some Nrf2 responsive genes (several metallothioneins, HMOX, G6PD, OSGIN1 and GPX2) were highly up regulated. Members of the selenoproteins were also differentially expressed. Our findings indicate that exposure to high concentration of AuNPs (5nm) induces metal exposure, oxidative stress signaling pathways, and might influence selenium homeostasis. Some of detected cellular responses might be explored as potential enhancers of anti-cancer properties of AuNPs based nanomedicines.

H2AX phosphorylation in A549 cells induced by the bulky and stable DNA adducts of benzo[a]pyrene and dibenzo[a,l]pyrene diol epoxides.

Early events in the cellular response to DNA damage, such as double strand breaks, rely on lesion recognition and activation of proteins involved in maintenance of genomic stability. One important component of this process is the phosphorylation of the histone variant H2AX. To investigate factors explaining the variation in carcinogenic potency between different categories of polycyclic aromatic hydrocarbons (PAHs), we have studied the phosphorylation of H2AX (H2AXgamma). A549 cells were exposed to benzo[a]pyrene diol epoxide [(+)-anti-BPDE] (a bay-region PAH) and dibenzo[a,l]pyrene diol epoxide [(-)-anti-DBPDE] (a fjord-region PAH) and H2AXgamma was studied using immunocytochemistry and Western blot. Hydrogen peroxide (H(2)O(2)) was used to induce oxidative DNA damage and strand breaks. As showed with single cell gel electrophoresis, neither of the diol epoxides resulted in DNA strand breaks relative to H(2)O(2). Visualisation of H2AXgamma formation demonstrated that the proportion of cells exhibiting H2AXgamma staining at 1h differed between BPDE, 40% followed by a decline, and DBPDE, <10% followed by an increase. With H(2)O(2) treatment, almost all cells demonstrated H2AXgamma at 1h. Western blot analysis of the H2AXgamma formation also showed concentration and time-dependent response patterns. The kinetics of H2AXgamma formation correlated with the previously observed kinetics of elimination of BPDE and DBPDE adducts. Thus, the extent of H2AXgamma formation and persistence was related to both the number of adducts and their structural features.

Efficient 5' cap-dependent RNA purification : use in identifying and studying subsets of RNA.

Microarray-based screening technologies have revealed a larger than expected diversity of gene expression profiles for many cells, tissues, and organisms. The complexity of RNA species, defined by their molecular structure, represents a major new development in biology. RNA not only carries genetic information in the form of templates and components of the translational machinery for protein synthesis but also directly regulates gene expression as exemplified by micro-RNAs (miRNAs). Recent evidence has demonstrated that 5' capped and 3' polyadenylated ends are not restricted to mRNAs, but that they are also present in precursors of both miRNAs and some antisense RNA transcripts. In addition, as many as 40% of transcribed RNAs may lack 3' poly(A) ends. In concert with the presence of a 5' cap (m7 GpppN), the length of the 3' poly(A) end plays a critical role in determining the translational efficiency, stability, and the cellular distribution of a specific mRNA. RNAs with short or lacking 3' poly(A) ends, that escape isolation and amplification with oligo(dT)-based methods, provide a challenge in RNA biology and gene expression studies. To circumvent the limitations of 3' poly(A)-dependent RNA isolation methods, we developed an efficient RNA purification system that binds the 5' cap of RNA with a high-affinity variant of the cap-binding protein eIF4E. This system can be used in differential selection approaches to isolate subsets of RNAs, including those with short 3' poly(A) ends that are likely targets of post-transcriptional regulation of gene expression. The length of the 3' poly(A) ends can be defined using a rapid polymerase chain reaction (PCR)- based approach.

Genomic analysis suggests higher susceptibility of children to air pollution.

Differences in biological responses to exposure to hazardous airborne substances between children and adults have been reported, suggesting children to be more susceptible. Aim of this study was to improve our understanding of differences in susceptibility in cancer risk associated with air pollution by comparing genome-wide gene expression profiles in peripheral blood of children and their parents. Gene expression analysis was performed in blood from children and parents living in two different regions in the Czech Republic with different levels of air pollution. Data were analyzed by two different approaches: one method first selected significantly differentially expressed genes and analyzed these gene lists for overrepresented biological processes, whereas the other applied the T-profiler tool to directly perform pathway analyses on the total gene set without preselection of significantly modulated gene expressions. In addition, gene expressions in both children and adults were investigated for associations with micronuclei frequencies. Both analysis approaches returned considerably more genes or gene groups and pathways that significantly differed between children from both regions than between parents. Very little overlap was observed between children and adults. The two most important biological processes or molecular functions significantly modulated in children, but not in adults, are nucleosome and immune response related. Our study suggests differences between children and adults in relation to air pollution exposure at the transcriptome level. The findings underline the necessity of implementing environmental health policy measures specifically for protecting children's health.

Impact of the interaction between adenovirus E1A and CtBP on host cell gene expression.

In cell lines harbouring inducible adenovirus E1A genes, the cytotoxicity of wild type E1A was manifested by poor and subsiding expression of the E1A protein during prolonged induction. In contrast, cells expressing E1A deleted in the C-terminal binding protein (CtBP)-interaction domain (E1ADeltaCID) demonstrated high levels of expression for extended time. Microarray analyses of host cell gene expression demonstrated that approximately 70% of the regulated genes were increased upon E1A induction and that the majority of E1A-regulated genes were similarly regulated by wild type E1A and E1ADeltaCID. However, for 29 genes, regulation by wild type E1A and E1ADeltaCID were different. Consistent with the altered transforming capacity of E1A unable to bind CtBP, genes involved in tumour cell progression and growth suppression were found among the differently regulated genes. Moreover, promoter sequences of genes up regulated by wild type E1A and/or repressed by E1ADeltaCID demonstrated a higher prevalence of potential binding sites for the CtBP-targeted transcription factors Ets, Ikaros and/or partial differentialEF1/ZEB, suggesting that the failure to block CtBP-repression contributed to the "hyper-transforming" phenotype of E1ADeltaCID. Since E1ADeltaCID also specifically activated host cell gene expression, we find it likely that additional, possibly CtBP-independent, mechanisms contribute to the altered phenotype of E1ADeltaCID-expressing cells.

Hydrogen peroxide causes greater oxidation in cellular RNA than in DNA.

Human A549 lung epithelial cells were challenged with 18O-labeled hydrogen peroxide ([18O]-H2O2), the total RNA and DNA extracted in parallel, and analyzed for 18O-labeled 8-oxo-7,8-dihydroguanosine ([18O]-8-oxoGuo) and 8-oxo-7,8-dihydro-2'-deoxyguanosine ([18O]-8-oxodGuo) respectively, using high-performance liquid chromatography electrospray ionization tandem mass spectrometry (HPLC-MS/MS). [18O]-H2O2 exposure resulted in dose-response formation of both [18O]-8-oxoGuo and [18O]-8-oxodGuo and 18O-labeling of guanine in RNA was 14-25 times more common than in DNA. Kinetics of formation and subsequent removal of oxidized nucleic acids adducts were also monitored up to 24 h. The A549 showed slow turnover rates of adducts in RNA and DNA giving half-lives of approximately 12.5 h for [18O]-8-oxoGuo in RNA and 20.7 h for [18O]-8-oxodGuo in DNA, respectively.

Protein S-glutathionylation correlates to selective stress gene expression and cytoprotection.

During situations of oxidative stress phenotypic adaptation to altered redox state is achieved by changes in expression of selected genes. The mechanisms regulating this may involve reversible S-glutathionylation of cellular proteins. In this study we compared and contrasted changes in gene expression patterns in human type II lung epithelial A549 cells and human endothelial ECV304 cells in correlation to glutathione oxidation and the formation of glutathione-protein mixed disulphides, after exposure to subtoxic levels of hydrogen peroxide, formed in the medium by addition of glucose oxidase, or the thiol oxidant diamide. Both the number of specific mRNAs and their levels of induction were grossly correlated to the degree of S-glutathionylation of cellular protein. Thus, diamide induced the expression of a variety of protein and DNA chaperones and transcriptional regulators, particularly in ECV304 cells. On the other hand, the peroxide failed to induce many of these species, in association with only minimal disturbances to glutathione homeostasis. The induction of the chaperone responses at the level of mRNA was clearly shown to translate into a more resistant morphological phenotype in response to both heat shock and oxidative stress induced by the DNA-damaging pro-oxidant potassium bromate.