Optimised laser microdissection of the human ocular surface epithelial regions for microarray studies

Background The most important challenge of performing insitu transcriptional profiling of the human ocular surface epithelial regions is obtaining samples in sufficient amounts, without contamination from adjacent tissue, as the region of interest is microscopic and closely apposed to other tissues regions. We have effectively collected ocular surface (OS) epithelial tissue samples from the Limbal Epithelial Crypt (LEC), limbus, cornea and conjunctiva of post-mortem cadaver eyes with laser microdissection (LMD) technique for gene expression studies with spotted oligonucleotide microarrays and Gene 1.0 ST arrays. Methods Human donor eyes (4 pairs for spotted oligonucleotide microarrays, 3 pairs for Gene 1.0 ST arrays) consented for research were included in this study with due ethical approval of the Nottingham Research Ethics Committee. Eye retrieval was performed within 36 hours of post-mortem period. The dissected corneoscleral buttons were immersed in OCT media and frozen in liquid nitrogen and stored at −80°C till further use. Microscopic tissue sections of interest were taken on PALM slides and stained with Toluidine Blue for laser microdissection with PALM microbeam systems. Optimisation of the laser microdissection technique was crucial for efficient and cost effective sample collection. Results The starting concentration of RNA as stipulated by the protocol of microarray platforms was taken as the cut-off concentration of RNA samples in our studies. The area of LMD tissue processed for spotted oligonucleotide microarray study ranged from 86,253 μm2 in LEC to 392,887 μm2 in LEC stroma. The RNA concentration of the LMD samples ranged from 22 to 92 pg/μl. The recommended starting concentration of the RNA samples used for Gene 1.0 ST arrays was 6 ng/5 μl. To achieve the desired RNA concentration the area of ocular surface epithelial tissue sample processed for the Gene 1.0 ST array experiments was approximately 100,0000 μm2 to 130,0000 μm2. RNA concentration of these samples ranged from 10.88 ng/12 μl to 25.8 ng/12 μl, with the RNA integrity numbers (RIN) for these samples from 3.3 to 7.9. RNA samples with RIN values below 2, that had failed to amplify satisfactorily were discarded. Conclusions The optimised protocol for sample collection and laser microdissection improved the RNA yield of the insitu ocular surface epithelial regions for effective microarray studies on spotted oligonucleotide and affymetrix platforms

Long-term (trophic) purinergic signalling: purinoceptors control cell proliferation, differentiation and death

The purinergic signalling system, which uses purines and pyrimidines as chemical transmitters, and purinoceptors as effectors, is deeply rooted in evolution and development and is a pivotal factor in cell communication. The ATP and its derivatives function as a 'danger signal' in the most primitive forms of life. Purinoceptors are extraordinarily widely distributed in all cell types and tissues and they are involved in the regulation of an even more extraordinary number of biological processes. In addition to fast purinergic signalling in neurotransmission, neuromodulation and secretion, there is long-term (trophic) purinergic signalling involving cell proliferation, differentiation, motility and death in the development and regeneration of most systems of the body. In this article, we focus on the latter in the immune/defence system, in stratified epithelia in visceral organs and skin, embryological development, bone formation and resorption, as well as in cancer. Cell Death and Disease (2010) 1, e9; doi:10.1038/cddis.2009.11; published online 14 January 201

Non-Invasive In Vivo Imaging of Tumor-Associated CD133/Prominin

detection of cancer stem cells is of great importance. detection of CD133/prominin, a cancer stem cell surface marker for a variety of tumor entities. The CD133-specific monoclonal antibody AC133.1 was used for quantitative fluorescence-based optical imaging of mouse xenograft models based on isogenic pairs of CD133 positive and negative cell lines. A first set consisted of wild-type U251 glioblastoma cells, which do not express CD133, and lentivirally transduced CD133-overexpressing U251 cells. A second set made use of HCT116 colon carcinoma cells, which uniformly express CD133 at levels comparable to primary glioblastoma stem cells, and a CD133-negative HCT116 derivative. Not surprisingly, visualization and quantification of CD133 in overexpressing U251 xenografts was successful; more importantly, however, significant differences were also found in matched HCT116 xenograft pairs, despite the lower CD133 expression levels. The binding of i.v.-injected AC133.1 antibodies to CD133 positive, but not negative, tumor cells isolated from xenografts was confirmed by flow cytometry. imaging of tumor-associated CD133 is feasible and that CD133 antibody-based tumor targeting is efficient. This should facilitate developing clinically applicable cancer stem cell imaging methods and CD133 antibody-based therapeutics

The Preclinical Natural History of Serous Ovarian Cancer: Defining the Target for Early Detection

Pat Brown and colleagues carry out a modeling study and define what properties a biomarker-based screening test would require in order to be clinically useful

Phagosomal Rupture by Mycobacterium tuberculosis Results in Toxicity and Host Cell Death

Survival within macrophages is a central feature of Mycobacterium tuberculosis pathogenesis. Despite significant advances in identifying new immunological parameters associated with mycobacterial disease, some basic questions on the intracellular fate of the causative agent of human tuberculosis in antigen-presenting cells are still under debate. To get novel insights into this matter, we used a single-cell fluorescence resonance energy transfer (FRET)-based method to investigate the potential cytosolic access of M. tuberculosis and the resulting cellular consequences in an unbiased, quantitative way. Analysis of thousands of THP-1 macrophages infected with selected wild-type or mutant strains of the M. tuberculosis complex unambiguously showed that M. tuberculosis induced a change in the FRET signal after 3 to 4 days of infection, indicating phagolysosomal rupture and cytosolic access. These effects were not seen for the strains M. tuberculosisΔRD1 or BCG, both lacking the ESX-1 secreted protein ESAT-6, which reportedly shows membrane-lysing properties. Complementation of these strains with the ESX-1 secretion system of M. tuberculosis restored the ability to cause phagolysosomal rupture. In addition, control experiments with the fish pathogen Mycobacterium marinum showed phagolysosomal translocation only for ESX-1 intact strains, further validating our experimental approach. Most importantly, for M. tuberculosis as well as for M. marinum we observed that phagolysosomal rupture was followed by necrotic cell death of the infected macrophages, whereas ESX-1 deletion- or truncation-mutants that remained enclosed within phagolysosomal compartments did not induce such cytotoxicity. Hence, we provide a novel mechanism how ESX-1 competent, virulent M. tuberculosis and M. marinum strains induce host cell death and thereby escape innate host defenses and favor their spread to new cells. In this respect, our results also open new research directions in relation with the extracellular localization of M. tuberculosis inside necrotic lesions that can now be tackled from a completely new perspective

Socioeconomic inequalities in the use of outpatient services in Brazil according to health care need: evidence from the World Health Survey

<p>Abstract</p> <p>Background</p> <p>The Brazilian health system is founded on the principle of equity, meaning provision of equal care for equal needs. However, little is known about the impact of health policies in narrowing socioeconomic health inequalities. Using data from the Brazilian World Health Survey, this paper addresses socioeconomic inequalities in the use of outpatient services according to intensity of need.</p> <p>Methods</p> <p>A three-stage cluster sampling was used to select 5000 adults (18 years and over). The non-response rate was 24.7% and calibration of the natural expansion factors was necessary to obtain the demographic structure of the Brazilian population. Utilization was established by use of outpatient services in the 12 months prior to the interview. Socioeconomic inequalities were analyzed by logistic regression models using years of schooling and private health insurance as independent variables, and controlling by age and sex. Effects of the socioeconomic variables on health services utilization were further analyzed according to self-rated health (good, fair and poor), considered as an indicator of intensity of health care need.</p> <p>Results</p> <p>Among the 5000 respondents, 63.4% used an outpatient service in the year preceding the survey. The association of health services utilization and self-rated health was significant (p < 0.001). Regarding socioeconomic inequalities, the less educated used health services less frequently, despite presenting worse health conditions. Highly significant effects were found for both socioeconomic variables, years of schooling (p < 0.001) and private health insurance (p < 0.00), after controlling for age and sex. Stratifying by self-rated health, the effects of both socioeconomic variables were significant among those with good health status, but not statistically significant among those with poor self-rated health.</p> <p>Conclusions</p> <p>The analysis showed that the social gradient in outpatient services utilization decreases as the need is more intense. Among individuals with good self-rated health, possible explanations for the inequality are the lower use of preventive services and unequal supply of health services among the socially disadvantaged groups, or excessive use of health services by the wealthy. On the other hand, our results indicate an adequate performance of the Brazilian health system in narrowing socioeconomic inequalities in health in the most serious situations of need.</p

Sequential Adaptive Mutations Enhance Efficient Vector Switching by Chikungunya Virus and Its Epidemic Emergence

The adaptation of Chikungunya virus (CHIKV) to a new vector, the Aedes albopictus mosquito, is a major factor contributing to its ongoing re-emergence in a series of large-scale epidemics of arthritic disease in many parts of the world since 2004. Although the initial step of CHIKV adaptation to A. albopictus was determined to involve an A226V amino acid substitution in the E1 envelope glycoprotein that first arose in 2005, little attention has been paid to subsequent CHIKV evolution after this adaptive mutation was convergently selected in several geographic locations. To determine whether selection of second-step adaptive mutations in CHIKV or other arthropod-borne viruses occurs in nature, we tested the effect of an additional envelope glycoprotein amino acid change identified in Kerala, India in 2009. This substitution, E2-L210Q, caused a significant increase in the ability of CHIKV to develop a disseminated infection in A. albopictus, but had no effect on CHIKV fitness in the alternative mosquito vector, A. aegypti, or in vertebrate cell lines. Using infectious viruses or virus-like replicon particles expressing the E2-210Q and E2-210L residues, we determined that E2-L210Q acts primarily at the level of infection of A. albopictus midgut epithelial cells. In addition, we observed that the initial adaptive substitution, E1-A226V, had a significantly stronger effect on CHIKV fitness in A. albopictus than E2-L210Q, thus explaining the observed time differences required for selective sweeps of these mutations in nature. These results indicate that the continuous CHIKV circulation in an A. albopictus-human cycle since 2005 has resulted in the selection of an additional, second-step mutation that may facilitate even more efficient virus circulation and persistence in endemic areas, further increasing the risk of more severe and expanded CHIK epidemics

The Context of Current Content Analysis of Gender Roles: An Introduction to a Special Issue

The aim of this paper is to provide context for the quantitative content analyses of gender roles that are to be included in both parts of this special issue. First, a timeline of historical uses of the content analysis methodology is presented. Second, research objectives that frequently drive content analysis of gender roles are described; these include: to support feminist claims, to compare media with real life, to predict effects on audiences, and to detect effects of media producers on content. Third, previous content analyses published in Sex Roles and other gender-focused journals are reviewed and categorized in terms of medium, genre, time span, gender, and nationality. Finally, contributions of each of the articles in this special issue are outlined

The role of the myosin ATPase activity in adaptive thermogenesis by skeletal muscle

Resting skeletal muscle is a major contributor to adaptive thermogenesis, i.e., the thermogenesis that changes in response to exposure to cold or to overfeeding. The identification of the “furnace” that is responsible for increased heat generation in resting muscle has been the subject of a number of investigations. A new state of myosin, the super relaxed state (SRX), with a very slow ATP turnover rate has recently been observed in skeletal muscle (Stewart et al. in Proc Natl Acad Sci USA 107:430–435, 2010). Inhibition of the myosin ATPase activity in the SRX was suggested to be caused by binding of the myosin head to the core of the thick filament in a structural motif identified earlier by electron microscopy. To be compatible with the basal metabolic rate observed in vivo for resting muscle, most myosin heads would have to be in the SRX. Modulation of the population of this state, relative to the normal relaxed state, was proposed to be a major contributor to adaptive thermogenesis in resting muscle. Transfer of only 20% of myosin heads from the SRX into the normal relaxed state would cause muscle thermogenesis to double. Phosphorylation of the myosin regulatory light chain was shown to transfer myosin heads from the SRX into the relaxed state, which would increase thermogenesis. In particular, thermogenesis by myosin has been proposed to play a role in the dissipation of calories during overfeeding. Up-regulation of muscle thermogenesis by pharmaceuticals that target the SRX would provide new approaches to the treatment of obesity or high blood sugar levels