Targeted knock-down of miR21 primary transcripts using snoMEN vectors induces apoptosis in human cancer cell lines

We have previously reported an antisense technology, 'snoMEN vectors', for targeted knock-down of protein coding mRNAs using human snoRNAs manipulated to contain short regions of sequence complementarity with the mRNA target. Here we characterise the use of snoMEN vectors to target the knock-down of micro RNA primary transcripts. We document the specific knock-down of miR21 in HeLa cells using plasmid vectors expressing miR21-targeted snoMEN RNAs and show this induces apoptosis. Knock-down is dependent on the presence of complementary sequences in the snoMEN vector and the induction of apoptosis can be suppressed by over-expression of miR21. Furthermore, we have also developed lentiviral vectors for delivery of snoMEN RNAs and show this increases the efficiency of vector transduction in many human cell lines that are difficult to transfect with plasmid vectors. Transduction of lentiviral vectors expressing snoMEN targeted to pri-miR21 induces apoptosis in human lung adenocarcinoma cells, which express high levels of miR21, but not in human primary cells. We show that snoMEN-mediated suppression of miRNA expression is prevented by siRNA knock-down of Ago2, but not by knock-down of Ago1 or Upf1. snoMEN RNAs colocalise with Ago2 in cell nuclei and nucleoli and can be co-immunoprecipitated from nuclear extracts by antibodies specific for Ago2

An instability criterion for nonlinear standing waves on nonzero backgrounds

A nonlinear Schr\"odinger equation with repulsive (defocusing) nonlinearity is considered. As an example, a system with a spatially varying coefficient of the nonlinear term is studied. The nonlinearity is chosen to be repelling except on a finite interval. Localized standing wave solutions on a non-zero background, e.g., dark solitons trapped by the inhomogeneity, are identified and studied. A novel instability criterion for such states is established through a topological argument. This allows instability to be determined quickly in many cases by considering simple geometric properties of the standing waves as viewed in the composite phase plane. Numerical calculations accompany the analytical results.Comment: 20 pages, 11 figure

Qualitative description – the poor cousin of health research?

<p>Abstract</p> <p>Background</p> <p>The knowledge and use of qualitative description as a qualitative research approach in health services research is limited.</p> <p>The aim of this article is to discuss the potential benefits of a qualitative descriptive approach, to identify its strengths and weaknesses and to provide examples of use.</p> <p>Discussion</p> <p>Qualitative description is a useful qualitative method in much medical research if you keep the limitations of the approach in mind. It is especially relevant in mixed method research, in questionnaire development and in research projects aiming to gain firsthand knowledge of patients', relatives' or professionals' experiences with a particular topic. Another great advantage of the method is that it is suitable if time or resources are limited.</p> <p>Summary</p> <p>As a consequence of the growth in qualitative research in the health sciences, researchers sometimes feel obliged to designate their work as phenomenology, grounded theory, ethnography or a narrative study when in fact it is not. Qualitative description might be a useful alternative approach to consider.</p

Regulatory subunits of PKA define an axis of cellular proliferation/differentiation in ovarian cancer cells

<p>Abstract</p> <p>Background</p> <p>The regulatory subunit of cAMP-dependent protein kinase (PKA) exists in two isoforms, RI and RII, which distinguish the PKA isozymes, type I (PKA-I) and type II (PKA-II). Evidence obtained from a variety of different experimental approaches has shown that the relative levels of type I and type II PKA in cells can play a major role in determining the balance between cell growth and differentiation. In order to characterize the effect of PKA type I and type II regulatory subunits on gene transcription at a global level, the PKA regulatory subunit genes for RIα and RIIβ were stably transfected into cells of the ovarian cancer cell line (OVCAR8).</p> <p>Results</p> <p>RIα transfected cells exhibit hyper-proliferative growth and RIIβ transfected cells revert to a relatively quiescent state. Profiling by microarray revealed equally profound changes in gene expression between RIα, RIIβ, and parental OVCAR cells. Genes specifically up-regulated in RIα cells were highly enriched for pathways involved in cell growth while genes up-regulated in RIIβ cells were enriched for pathways involved in differentiation. A large group of genes (~3600) was regulated along an axis of proliferation/differentiation between RIα, parental, and RIIβ cells. RIα/wt and RIIβ/wt gene regulation was shown by two separate and distinct gene set analytical methods to be strongly cross-correlated with a generic model of cellular differentiation.</p> <p>Conclusion</p> <p>Overexpression of PKA regulatory subunits in an ovarian cancer cell line dramatically influences the cell phenotype. The proliferation phenotype is strongly correlated with recently identified clinical biomarkers predictive of poor prognosis in ovarian cancer suggesting a possible pivotal role for PKA regulation in disease progression.</p

Sex and sport: chlamydia screening in rural sporting clubs

BACKGROUND: Chlamydia trachomatis is the most common notifiable disease in Australia, mainly affecting those aged 15 to 29 years. Testing rates are low in Australia and considerably lower in rural areas, with access and confidentiality of sexual health services being problematic in rural and regional areas. This study aimed to determine the feasibility of establishing a pilot chlamydia testing outreach program among 16-25 year old males and females in rural Victoria (Australia) undertaken at local sporting clubs and to determine the prevalence of chlamydia and acceptability of the program in this population. METHODS: We aimed to recruit young people from the Loddon Mallee region of Victoria, Australia between May and September 2007. After a night of sporting practice, participants provided a first pass urine sample, completed a brief questionnaire regarding risk taking behaviour and were then provided with condoms and health promotion materials about sexually transmitted infections (STIs). Those positive for chlamydia were managed by telephone consultation with a practitioner from Melbourne Sexual Health Centre. RESULTS: A total of 709 young people participated (77% male, 23% female), 77% being sexually active. All provided a urine sample and completed the questionnaire. Participation rate on recruitment nights was over 95%. Overall chlamydia prevalence in those sexually active was 5.1% (95%CI: 3.4-7.3), 7.4% in females (95%CI: 3.5-13.6) and 4.5% in males (95%CI: 2.7-6.9). CONCLUSION: Sporting clubs represent a feasible, acceptable and innovative community based setting to screen, treat and educate young people in a rural and regional setting, especially for males

Physical and land-cover variables influence ant functional groups and species diversity along elevational gradients

Of particular importance in shaping species assemblages is the spatial heterogeneity of the environment. The aim of our study was to investigate the influence of spatial heterogeneity and environmental complexity on the distribution of ant functional groups and species diversity along altitudinal gradients in a temperate ecosystem (Pyrenees Mountains). During three summers, we sampled 20 sites distributed across two Pyrenean valleys ranging in altitude from 1,009 to 2,339 m by using pitfall traps and hand collection. The environment around each sampling points was characterized by using both physical and land-cover variables. We then used a self-organizing map algorithm (SOM, neural network) to detect and characterize the relationship between the spatial distribution of ant functional groups, species diversity, and the variables measured. The use of SOM allowed us to reduce the apparent complexity of the environment to five clusters that highlighted two main gradients: an altitudinal gradient and a gradient of environmental closure. The composition of ant functional groups and species diversity changed along both of these gradients and was differently affected by environmental variables. The SOM also allowed us to validate the contours of most ant functional groups by highlighting the response of these groups to the environmental and land-cover variables

Diabetes related risk factors did not explain the increased risk for urinary incontinence among women with diabetes. The Norwegian HUNT/EPINCONT study

<p>Abstract</p> <p>Background</p> <p>Previous studies have shown an association between diabetes mellitus (DM) and urinary incontinence (UI) in women, especially severe UI. The purpose of this study was to investigate whether diabetes related variables could explain this association.</p> <p>Methods</p> <p>The study is part of the EPINCONT study, which is based on the large Nord-Trøndelag Health Study 2 (HUNT 2), performed in the county of Nord-Trøndelag, Norway, during the years 1995 - 1997. Questions on diabetes and UI were answered by a total of 21 057 women aged 20 years and older. Of these 685 were identified as having diabetes, and thus comprise the population of our study. A variety of clinical and biochemical variables were recorded from the participants.</p> <p>Results</p> <p>Blood-glucose, HbA1c, albumine:creatinine ratio (ACR), duration of diabetes, diabetes treatment, type of diabetes, cholesterol and triglycerides did not significantly differ in women with and without UI in crude analyses. However, the diabetic women with UI had more hospitalizations during the last 12 months, more homecare, and a higher prevalence of angina and use of oestrogene treatment (both local and oral/patch). After adjusting for age, BMI, parity and smoking, there were statistically significant associations between any UI and angina (OR 1.89; 95% CI: 1.22 - 2.93), homecare (OR 1.72; 95% CI: 1.02 - 2.89), and hospitalization during the last 12 months (OR 1.67; 95% CI: 1.18 - 2.38). In adjusted analyses severe UI was also significantly associated with the same variables, and also with diabetes drug treatment (OR 2.10; 95% CI: 1.07 - 4.10) and stroke (OR 2.47; 95% CI: 1.09 - 5.59).</p> <p>Conclusion</p> <p>No single diabetes related risk factor seems to explain the increased risk for UI among women with diabetes. However, we found associations between UI and some clinical correlates of diabetes.</p

Identification of genes for normalization of real-time RT-PCR data in breast carcinomas

<p>Abstract</p> <p>Background</p> <p>Quantitative real-time RT-PCR (RT-qPCR) has become a valuable molecular technique in basic and translational biomedical research, and is emerging as an equally valuable clinical tool. Correlation of inter-sample values requires data normalization, which can be accomplished by various means, the most common of which is normalization to internal, stably expressed, reference genes. Recently, such traditionally utilized reference genes as GAPDH and B2M have been found to be regulated in various circumstances in different tissues, emphasizing the need to identify genes independent of factors influencing the tissue, and that are stably expressed within the experimental milieu. In this study, we identified genes for normalization of RT-qPCR data for invasive breast cancer (IBC), with special emphasis on estrogen receptor positive (ER+) IBC, but also examined their applicability to ER- IBC, normal breast tissue and breast cancer cell lines.</p> <p>Methods</p> <p>The reference genes investigated by qRT-PCR were RPLP0, TBP, PUM1, ACTB, GUS-B, ABL1, GAPDH and B2M. Biopsies of 18 surgically-excised tissue specimens (11 ER+ IBCs, 4 ER- IBCs, 3 normal breast tissues) and 3 ER+ cell lines were examined and the data analyzed by descriptive statistics, geNorm and NormFinder. In addition, the expression of selected reference genes in laser capture microdissected ER+ IBC cells were compared with that of whole-tissue.</p> <p>Results</p> <p>A group of 3 genes, TBP, RPLP0 and PUM1, were identified for both the combined group of human tissue samples (ER+ and ER- IBC and normal breast tissue) and for the invasive cancer samples (ER+ and ER- IBC) by GeNorm, where NormFinder consistently identified PUM1 at the single best gene for all sample combinations.</p> <p>Conclusion</p> <p>The reference genes of choice when performing RT-qPCR on normal and malignant breast specimens should be either the collected group of 3 genes (TBP, RPLP0 and PUM1) employed as an average, or PUM1 as a single gene.</p

Expression of DLK1 and MEG3 genes in porcine tissues during postnatal development

The Drosophila-like homolog 1 (DLK1), a transmembrane signal protein similar to other members of the Notch/Delta/Serrate family, regulates the differentiation process in many types of mammalian cells. Callipyge sheep and DLK1 knockout mice are excellent examples of a fundamental role of the gene encoding DLK1 in muscle growth and fat deposition. DLK1 is located within co-regulated imprinted clusters (the DLK1/DIO3 domain), along with other imprinted genes. Some of these, e.g. the RNA coding MEG3 gene, presumedly interfere with DLK1 transcription. The aim of our study was to analyze DLK1 and MEG3 gene expression in porcine tissues (muscle, liver, kidney, heart, brain stem) during postnatal development. The highest expression of both DLK1 and MEG3 variant 1 (MEG3 var.1) was observed in the brain-stem and muscles, whereas that of MEG3 variant 2 (MEG3var.2) was the most abundant in muscles and the heart. During development (between 60 and 210 days of age) expression of analyzed genes was down-regulated in all the tissues. An exception was the brain- stem, where there was no significant change in MEG3 (both variants) mRNA level, and relatively little decline (2-fold) in that of DLK1 transcription. This may indicate a distinct function of the DLK1 gene in the brain-stem, when compared with other tissues