Expression of the RNA helicase DDX3 and the hypoxia response in breast cancer

<p>Aims: DDX3 is an RNA helicase that has antiapoptotic properties, and promotes proliferation and transformation. In addition, DDX3 was shown to be a direct downstream target of HIF-1α (the master regulatory of the hypoxia response) in breast cancer cell lines. However, the relation between DDX3 and hypoxia has not been addressed in human tumors. In this paper, we studied the relation between DDX3 and the hypoxic responsive proteins in human breast cancer.</p> <p>Methods and Results: DDX3 expression was investigated by immunohistochemistry in breast cancer in comparison with hypoxia related proteins HIF-1α, GLUT1, CAIX, EGFR, HER2, Akt1, FOXO4, p53, ERα, COMMD1, FER kinase, PIN1, E-cadherin, p21, p27, Transferrin receptor, FOXO3A, c-Met and Notch1. DDX3 was overexpressed in 127 of 366 breast cancer patients, and was correlated with overexpression of HIF-1α and its downstream genes CAIX and GLUT1. Moreover, DDX3 expression correlated with hypoxia-related proteins EGFR, HER2, FOXO4, ERα and c-Met in a HIF-1α dependent fashion, and with COMMD1, FER kinase, Akt1, E-cadherin, TfR and FOXO3A independent of HIF-1α.</p> <p>Conclusions: In invasive breast cancer, expression of DDX3 was correlated with overexpression of HIF-1α and many other hypoxia related proteins, pointing to a distinct role for DDX3 under hypoxic conditions and supporting the oncogenic role of DDX3 which could have clinical implication for current development of DDX3 inhibitors.</p&gt

Homeopathy – what are the active ingredients? An exploratory study using the UK Medical Research Council's framework for the evaluation of complex interventions

BACKGROUND: Research in homeopathy has traditionally addressed itself to defining the effectiveness of homeopathic potencies in comparison to placebo medication. There is now increasing awareness that the homeopathic consultation is in itself a therapeutic intervention working independently or synergistically with the prescribed remedy. Our objective was to identify and evalute potential "active ingredients" of the homeopathic approach as a whole, in a prospective formal case series, which draws on actual consultation data, and is based on the MRC framework for the evaluation of complex interventions. METHODS: Following on from a theoretical review of how homeopathic care might mediate its effects, 18 patients were prospectively recruited to a case series based at Bristol Homeopathic Hospital. Patients, who lived with one of three index conditions, were interviewed before and after a five visit "package of care". All consultations were recorded and transcribed verbatim. Additional data, including generic and condition-specific questionnaires, artwork and "significant other" reports were collected. Textual data was subject to thematic analysis and triangulated with other sources. RESULTS: We judged that around one third of patients had experienced a major improvement in their health over the study period, a third had some improvement and a third had no improvement. Putative active ingredients included the patients' "openness to the mind-body connection", consultational empathy, in-depth enquiry into bodily complaints, disclosure, the remedy matching process and, potentially, the homeopathic remedies themselves. CONCLUSION: This study has has identified, using primary consultation and other data, a range of factors that might account for the effectiveness of homeopathic care. Some of these, such as empathy, are non-specific. Others, such as the remedy matching process, are specific to homeopathy. These findings counsel against the use of placebo-controlled RCT designs in which both arms would potentially be receiving specific active ingredients. Future research in homeopathy should focus on pragmatic trials and seek to confirm or refute the therapeutic role of constructs such as patient "openness", disclosure and homeopathicity

Novel ATP-Independent RNA Annealing Activity of the Dengue Virus NS3 Helicase

The flavivirus nonstructural protein 3 (NS3) bears multiple enzymatic activities and represents an attractive target for antiviral intervention. NS3 contains the viral serine protease at the N-terminus and ATPase, RTPase, and helicase activities at the C-terminus. These activities are essential for viral replication; however, the biological role of RNA remodeling by NS3 helicase during the viral life cycle is still unclear. Secondary and tertiary RNA structures present in the viral genome are crucial for viral replication. Here, we used the NS3 protein from dengue virus to investigate functions of NS3 associated to changes in RNA structures. Using different NS3 variants, we characterized a domain spanning residues 171 to 618 that displays ATPase and RNA unwinding activities similar to those observed for the full-length protein. Interestingly, we found that, besides the RNA unwinding activity, dengue virus NS3 greatly accelerates annealing of complementary RNA strands with viral or non-viral sequences. This new activity was found to be ATP-independent. It was determined that a mutated NS3 lacking ATPase activity retained full-RNA annealing activity. Using an ATP regeneration system and different ATP concentrations, we observed that NS3 establishes an ATP-dependent steady state between RNA unwinding and annealing, allowing modulation of the two opposing activities of this enzyme through ATP concentration. In addition, we observed that NS3 enhanced RNA-RNA interactions between molecules representing the ends of the viral genome that are known to be necessary for viral RNA synthesis. We propose that, according to the ATP availability, NS3 could function regulating the folding or unfolding of viral RNA structures

Esperança de vida ao nascer: impacto das variações na mortalidade por idade e causas de morte no Município de Campinas, São Paulo, Brasil Life expectancy at birth: impact of variation in mortality by age group and cause of death in Campinas, São Paulo State, Brazil

O objetivo do estudo foi examinar o impacto das mudanças na mortalidade por idades e causas de morte sobre o aumento da esperança de vida ao nascer no Município de Campinas, São Paulo, Brasil, entre 1991, 2000 e 2005. Foram construídas tábuas de vida. O método de Pollard foi aplicado para estimar as contribuições das idades e causas de morte na variação da longevidade. O grupo etário de 0-1 ano foi o que mais contribuiu com o aumento da vida média masculina (31,1%) e feminina (22,9%) em 1991/2000. Entre 2000 e 2005, as idades de 15-44 anos responderam por 79% do ganho masculino. A maior contribuição entre 1991 e 2000 foi gerada pelas doenças cardiovasculares (66,1% entre os homens e 43,5% entre as mulheres). As causas externas subtraíram 1,1 ano entre os homens. Entre 2000 e 2005, com a queda da mortalidade por estas causas, a esperança de vida masculina aumentou em 2,3 anos. As neoplasias provocaram redução de 0,11 ano para homens e 0,15 ano para mulheres. Estes resultados podem auxiliar na orientação de políticas públicas de saúde para redução da mortalidade e aumento da esperança de vida ao nascer.<br>This study investigated the impact of variation in mortality by age group and cause of death on gains in life expectancy at birth in the city of Campinas, São Paulo State, Brazil, in 1991, 2000, and 2005. Life tables were constructed. Pollard's method was used to estimate the contributions by age group and cause of death on gains in life expectancy. In 1991-2000, the age group that most contributed was 0-1 year (31.1% for males and 22.9% for females). In 2000-2005, 79% of the gain for males was the result of mortality improvements in the 15-44-year bracket. Cardiovascular diseases made the largest contribution in 1991-2000 (66.1% for males and 43.5% for females). A loss in longevity was seen in men (1.1 year) resulting from increased mortality from external causes. In 2000-2005, the substantial gain (2.3 year) in male life expectancy was due to a reduction in mortality from external causes. Neoplasms had a negative effect on the gain (0.11 year for males and 0.15 for females). These findings should help support public health policies to reduce mortality risks and increase life expectancy

Real-time fluorescence assays to monitor duplex unwinding and ATPase activities of helicases.

Many physiological functions of helicases are dependent on their ability to unwind nucleic acid duplexes in an ATP-dependent fashion. Determining the kinetic frameworks of these processes is crucial to understanding how these proteins function. We recently developed a fluorescence assay to monitor RNA duplex unwinding by DEAD-box helicases in real time. In this assay, two fluorescently modified short reporter oligonucleotides are annealed to an unmodified RNA loading strand of any length so that the fluorescent moieties of the two reporters find themselves in close proximity to each other and fluorescence is quenched. One reporter is modified with cyanine 3 (Cy3), whereas the other is modified with a spectrally paired black-hole quencher (BHQ). As the helicase unwinds the loading strand, the enzyme displaces the Cy3-modified reporter, which will bind to a capture or competitor DNA strand, permanently separating it from the BHQ-modified reporter. Complete separation of the Cy3-modified reporter strand is thus detected as an increase in total fluorescence. This assay is compatible with reagentless biosensors to monitor ATPase activity so that the coupling between ATP hydrolysis and duplex unwinding can be determined. With the protocol described, obtaining data and analyzing results of unwinding and ATPase assays takes ∼4 h