Can behavioral interventions be too salient? Evidence from traffic safety messages.

Although behavioral interventions are designed to seize attention, little consideration has been given to the costs of doing so. We estimated these costs in the context of a safety campaign that, to encourage safe driving, displays traffic fatality counts on highway dynamic message signs for 1 week each month. We found that crashes increase statewide during campaign weeks, which is inconsistent with any benefits. Furthermore, these effects do not persist beyond campaign weeks. Our results show that behavioral interventions, particularly negatively framed ones, can be too salient, crowding out more important considerations and causing interventions to backfire-with costly consequences.

ZEB1 coordinately regulates laminin-332 and {beta}4 integrin expression altering the invasive phenotype of prostate cancer cells.

Metastasis involves the invasion of cancer cells across both the extracellular matrix and cellular barriers, and an evolving theme is that epithelial-to-mesenchymal transition (EMT) may mediate invasive cellular behavior. Previously, we isolated and analyzed a subpopulation of PC-3 prostate cancer cells, TEM4-18, and found that these cells both invaded an endothelial barrier more efficiently and exhibited enhanced metastatic colonization in vivo. Transendothelial migration of these cells depended on expression of ZEB1, a known regulator of EMT. Surprisingly, these cells were much less invasive than parental PC-3 cells in assays that involve matrix barriers. Here, we report that TEM4-18 cells express significantly reduced levels of two subunits of laminin-332 (ß3 and γ2) and that exogenous laminin-332, or co-culture with laminin-332-expressing cells, rescues the in vitro invasion phenotype in these cells. Stable knockdown of ZEB1 in prostate cancer cells up-regulated LAMC2 and ITGB4 mRNA and protein and resulted in a concomitant increase in Transwell migration. Using chromatin immunoprecipitation (ChIP), we show that ZEB1 directly interacts with the promoters of LAMC2 and ITGB4. These results provide a novel molecular basis for reduced laminin-332 observed in clinical prostate cancer specimens and demonstrate a context-dependent role for EMT in invasive cellular behavior.

Role of viral splicing elements and cellular RNA binding proteins in regulation of HIV-1 alternative RNA splicing.

In HIV-1 infected cells, over 40 different mRNA species are produced by alternative splicing of the single HIV-1 primary RNA transcript. In addition, approximately half of the HIV-1 primary RNA transcripts are not spliced and are exported to the cytoplasm where they serve as mRNA and as genomic RNA. In this article, we will review current knowledge of the mechanisms by which the HIV-1 alternative splicing is regulated. Several negatively and positively-acting cis-acting elements have been detected within the viral genome that repress or facilitate viral RNA splicing by binding to cellular proteins. These include exonic splicing silencers (ESS) and an intronic splicing silencer (ISS) that are selectively bound either by members of the hnRNP A/B family (hnRNPs A1, A1(B), A2, and B1) or by hnRNP H. Exonic splicing enhancers (ESE) are also present within the HIV-1 genome and are selectively bound by members of the SR protein family. ESS and ISS repression mediated by hnRNP A/B proteins occurs at early steps of splicing, prior to formation of pre-spliceosome complexes. Current models propose that ESS elements promote cooperative binding of hnRNP A/B proteins to the exon and prevent efficient binding of essential cellular splicing factors to the 3' splice site. SR proteins bound to ESE elements that are juxtaposed or overlapping ESS elements may counteract this inhibition. We will review data indicating the importance of the HIV-1 splicing elements and their cognate binding proteins for efficient virus replication. Differences in cis-acting splicing elements between the group M (major) and group O (outlier) HIV-1 strains will also be discussed. Finally we will review evidence suggesting the possibility that there may be changes in regulation of HIV-1 alternative splicing in infected human T cells, human macrophages and rodent cells.

A suboptimal 5' splice site downstream of HIV-1 splice site A1 is required for unspliced viral mRNA accumulation and efficient virus replication.

BACKGROUND: Inefficient alternative splicing of the human immunodeficiency virus type 1(HIV-1) primary RNA transcript results in greater than half of all viral mRNA remaining unspliced. Regulation of HIV-1 alternative splicing occurs through the presence of suboptimal viral 5' and 3' splice sites (5' and 3'ss), which are positively regulated by exonic splicing enhancers (ESE) and negatively regulated by exonic splicing silencers (ESS) and intronic splicing silencers (ISS). We previously showed that splicing at HIV-1 3'ss A2 is repressed by ESSV and enhanced by the downstream 5'ss D3 signal. Disruption of ESSV results in increased vpr mRNA accumulation and exon 3 inclusion, decreased accumulation of unspliced viral mRNA, and decreased virus production. RESULTS: Here we show that optimization of the 5'ss D2 signal results in increased splicing at the upstream 3'ss A1, increased inclusion of exon 2 into viral mRNA, decreased accumulation of unspliced viral mRNA, and decreased virus production. Virus production from the 5'ss D2 and ESSV mutants was rescued by transient expression of HIV-1 Gag and Pol. We further show that the increased inclusion of either exon 2 or 3 does not significantly affect the stability of viral mRNA but does result in an increase and decrease, respectively, in HIV-1 mRNA levels. The changes in viral mRNA levels directly correlate with changes in tat mRNA levels observed upon increased inclusion of exon 2 or 3. CONCLUSION: These results demonstrate that splicing at HIV-1 3'ss A1 is regulated by the strength of the downstream 5'ss signal and that suboptimal splicing at 3'ss A1 is necessary for virus replication. Furthermore, the replication defective phenotype resulting from increased splicing at 3'ss A1 is similar to the phenotype observed upon increased splicing at 3'ss A2. Further examination of the role of 5'ss D2 and D3 in the alternative splicing of 3'ss A1 and A2, respectively, is necessary to delineate a role for non-coding exon inclusion in HIV-1 replication.

Manganese superoxide dismutase depletion in murine hematopoietic stem cells perturbs iron homeostasis, globin switching, and epigenetic control in erythrocyte precursor cells.

Heme synthesis partially occurs in the mitochondrial matrix; thus there is a high probability that enzymes and intermediates important in the production of heme will be exposed to metabolic by-products including reactive oxygen species. In addition, the need for ferrous iron for heme production, Fe/S coordination, and other processes occurring in the mitochondrial matrix suggests that aberrant fluxes of reactive oxygen species in this compartment might perturb normal iron homeostasis. Manganese superoxide dismutase (Sod2) is an antioxidant enzyme that governs steady-state levels of the superoxide in the mitochondrial matrix. Using hematopoietic stem cell-specific conditional Sod2 knockout mice we observed increased superoxide concentrations in red cell progeny, which caused significant pathologies including impaired erythrocytes and decreased ferrochelatase activity. Animals lacking Sod2 expression in erythroid precursors also displayed extramedullary hematopoiesis and systemic iron redistribution. Additionally, the increase in superoxide flux in erythroid precursors caused abnormal gene regulation of hematopoietic transcription factors, globins, and iron-response genes. Moreover, the erythroid precursors also displayed evidence of global changes in histone posttranslational modifications, a likely cause of at least some of the aberrant gene expression noted. From a therapeutic translational perspective, mitochondrially targeted superoxide-scavenging antioxidants partially rescued the observed phenotype. Taken together, our findings illuminate the superoxide sensitivity of normal iron homeostasis in erythrocyte precursors and suggest a probable link between mitochondrial redox metabolism and epigenetic control of nuclear gene regulation during mammalian erythropoiesis.

Transgenic biosynthesis of trypanothione protects Escherichia coli from radiation-induced toxicity.

Trypanothione is a unique diglutathionyl-spermidine conjugate found in abundance in trypanosomes but not in other eukaryotes. Because trypanothione is a naturally occurring polyamine thiol reminiscent of the synthetic drug amifostine, it may be a useful protector against radiation and oxidative stress. For these reasons we hypothesized that trypanothione might serve as a radioprotective agent when produced in bacteria. To accomplish this objective, the trypanothione synthetase and reductase genes from T. cruzi were introduced into E. coli and their expression was verified by qPCR and immunoblotting. Trypanothione synthesis in bacteria, detected by HPLC, resulted in decreased intracellular levels of reactive oxygen species as determined by H(2)DCFDA oxidation. Moreover, E. coli genomic DNA was protected from radiation-induced DNA damage by 4.6-fold in the presence of trypanothione compared to control bacteria. Concordantly, the transgenic E. coli expressing trypanothione were 4.3-fold more resistant to killing by (137)Cs gamma radiation compared to E. coli devoid of trypanothione expression. Thus we have shown for the first time that E. coli can be genetically engineered to express the trypanothione biosynthetic pathway and produce trypanothione, which results in their radioresistance. These results warrant further research to explore the possibility of developing trypanothione as a novel radioprotective agent.

Transgenic biosynthesis of trypanothione protects Escherichia coli from radiation-induced toxicity

Trypanothione is a unique diglutathionyl-spermidine conjugate found in abundance in trypanosomes but not in other eukaryotes. Because trypanothione is a naturally occurring polyamine thiol reminiscent of the synthetic drug amifostine, it may be a useful protector against radiation and oxidative stress. For these reasons we hypothesized that trypanothione might serve as a radioprotective agent when produced in bacteria. To accomplish this objective, the trypanothione synthetase and reductase genes from T. cruzi were introduced into E. coli and their expression was verified by qPCR and immunoblotting. Trypanothione synthesis in bacteria, detected by HPLC, resulted in decreased intracellular levels of reactive oxygen species as determined by H(2)DCFDA oxidation. Moreover, E. coli genomic DNA was protected from radiation-induced DNA damage by 4.6-fold in the presence of trypanothione compared to control bacteria. Concordantly, the transgenic E. coli expressing trypanothione were 4.3-fold more resistant to killing by (137)Cs gamma radiation compared to E. coli devoid of trypanothione expression. Thus we have shown for the first time that E. coli can be genetically engineered to express the trypanothione biosynthetic pathway and produce trypanothione, which results in their radioresistance. These results warrant further research to explore the possibility of developing trypanothione as a novel radioprotective agent(AU)

An exonic splicing silencer downstream of the 3' splice site A2 is required for efficient human immunodeficiency virus type 1 replication.

Alternative splicing of the human immunodeficiency virus type 1 (HIV-1) genomic mRNA produces more than 40 unique viral mRNA species, of which more than half remain incompletely spliced within an HIV-1-infected cell. Regulation of splicing at HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation of splicing occurs through binding of cellular factors to cis-acting splicing regulatory elements. We have previously shown that splicing at HIV-1 3'ss A2, which produces vpr mRNA and promotes inclusion of HIV-1 exon 3, is repressed by the hnRNP A/B-dependent exonic splicing silencer ESSV. Here we show that ESSV activity downstream of 3'ss A2 is localized to a 16-nucleotide element within HIV-1 exon 3. HIV-1 replication was reduced by 95% when ESSV was inactivated by mutagenesis. Reduced replication was concomitant with increased inclusion of exon 3 within spliced viral mRNA and decreased accumulation of unspliced viral mRNA, resulting in decreased cell-associated p55 Gag. Prolonged culture of ESSV mutant viruses resulted in two independent second-site reversions disrupting the splice sites that define exon 3, 3'ss A2 and 5' splice site D3. Either of these changes restored both HIV-1 replication and regulated viral splicing. Therefore, inhibition of HIV-1 3'ss A2 splicing is necessary for HIV-1 replication.