Gene expression analysis in specific cell populations of bovine blastocysts using laser capture microdissection

Laser capture microdissection (LCM) has become a powerful technique for the isolation of specific cell populations from heterogeneous tissues for gene expression analysis. For standard laser capture microdissection procedures, snap freezing and cryosectioning is the optimal fixation and processing procedure. This sufficiently preserves RNA integrity for downstream molecular analysis. However, in the case of small sized samples, such as early stage embryos, snap freezing and cryosectioning is highly complex and frequently leads to the loss of valuable samples. In the present study, a protocol was optimized to specifically isolate the inner cell mass cells from bovine blastocysts with LCM on chemically fixated paraffin embedded embryos. Bovine blastocysts were fixed for 24h in a methacarn fixative (8 parts methanol, 1 part acetic acid), upon which they were embedded in 2% agarose. After paraffin embedding of the agarose blocks, serial sections of 10 µm were cut and adhered to glass slides. The slides were deparaffinized in xylene, stained with 0.1% cresyl violet in 85% ethanol, and dehydrated in xylene. Subsequently the trophectoderm cells and the inner cell mass cells were separately isolated using LCM. The RNA of the samples was isolated, and subjected to different tests to evaluate the purity of the samples and the quality of the RNA. Expression of Cytokeratin 18 which is only expressed in trophectoderm revealed that the isolated cells were highly pure. An amplicon length assay and a 3’-5’ assay revealed that the RNA quality was sufficient for gene expression analysis with RT-qPCR. In conclusion, LCM on blastocysts is well suited for gene expression analysis of specific cell populations, providing a novel strategy to investigate molecular pathways for pluripotency and cell fate development. This procedure is also compatible with micro-RNA profiling strategies, and the application for downstream transcriptome sequencing is currently being evaluated

Fluoxetine suppresses calcium signaling in human T lymphocytes through depletion of intracellular calcium stores

Selective serotonin reuptake inhibitors, such as fluoxetine, have recently been shown to exert anti-inflammatory and immunosuppressive effects. Although the effects on cytokine secretion, proliferation and viability of T lymphocytes have been extensively characterized, little is known about the mechanism behind these effects. It is well known that Ca2+ signaling is an important step in the signaling transduction pathway following T cell receptor activation. Therefore, we investigated if fluoxetine interferes with Ca2+ signaling in Jurkat T lymphocytes. Fluoxetine was found to suppress Ca2+ signaling in response to T cell receptor activation. Moreover, fluoxetine was found to deplete intracellular Ca2+ stores, thereby leaving less Ca2+ available for release upon IP3- and ryanodine-receptor activation. The Ca2+-modifying effects of fluoxetine are not related to its capability to block the serotonin transporter, as even a large excess of 5HT did not abolish the effects. In conclusion, these data show that fluoxetine decreases IP3- and ryanodine-receptor mediated Ca2+ release in Jurkat T lymphocytes, an effect likely to be at the basis of the observed immunosuppression

Moyo Vol. IX N 1

Durica, Paul. Editor\u27s Letter . 4. Fisher, D. The More you Resist, Babe, The More it Excites Me (Support for Beatty\u27s Bid) . 5. Ward, Luc. The haunting of Barney Hall. Mysteries of the Fourth Floor Revealed . 6. Million, Chris. Not Your Crocodile Dundee (Interview with Chet Snouffer) . 8. Curry, Kim. Phobia or Philia? Student Questions Prejudices Against Specters . 13. Hankinson, Tom. Supernatural is Super-Boring. Denison Sutdent Non-Plussed by Multiple Ghostly Experiences . 20. Anderson, Chris. Run, Rita, Run. Denison\u27s Marathon professor . 22. Durica, Paul. Night in Nine (Séance in Buxton Stirs More Than Spirits) . 10

Adapting to rising coastal flood risk in the EU under climate change

Around one third of the EU population lives within 50 km of the coast. Extreme sea levels in Europe could rise by as much as one meter or more by the end of this century. Without mitigation and adaptation measures, annual damages from coastal flooding in the EU-28 could increase sharply from €1.4 billion nowadays to almost €240 billion by 2100. Around 95% of these impacts could be avoided through moderate mitigation and by raising dykes where human settlements and economically important areas exist along the coastline. The extent to which adaptation can lessen the effects of coastal flooding and at what cost is sensitive to the investment strategy adopted.JRC.E.1-Disaster Risk Managemen

Compliance, hysteresis, and collapsibility of human small airways

We tested the hypothesis that airway wall dimensions are important determinants for the mechanical properties of airways. Lung tissue was obtained from 31 smokers with different degrees of chronic obstructive pulmonary disease (COPD) who were operated on for a solitary lung lesion. Segments of small airways (n = 35) were mounted on cannulas in an organ bath and inflated and deflated cyclically between +15 and -15 cm H(2)O. For each airway this was done at baseline, after methacholine, and after isoprenaline. Specific compliance (sCdyn), specific hysteresis (seta), and pressure at which the airways collapsed (Pcol) were calculated from each recording. Airway wall dimensions were measured morphometrically. Lung function parameters of airflow obstruction were correlated to sCdyn, seta, and Pcol. At baseline, after methacholine, and after isoprenaline sCdyn was 0.059, 0.052, and 0. 085 cm H(2)O(-)(1), seta was 13.5, 12.9, and 7.1%, and Pcol was -3.4, -3.5, and -1.9 cm H(2)O, respectively. Differences between sCdyn, seta, and Pcol after methacholine and after isoprenaline were highly significant (p < 0.001). Of all dimensions studied, smooth muscle area, but not total wall ar

Moyo Vol. VIII N 1

Durica, Paul Editor\u27s Letter . 4. Thackeray, Alex. Postcard from the sXe . 5. Ward, Luc. Gods & Monsters (Hook-Up at Church) . 6. Anshuman, Karan. The India Nobody Knows (Mysticism and Misconceptions Revealed) . 8. Clements, Nina and Betsy Falconer. God as One of Us: Diverse Faiths Thrive at Denison . 10. Million, Chris. Splendor in the Fall (First Year Love Bittersweet) . 16. Grindstaff, Michelle. Beer by Night, Bed by Morning . 17. Hart, Madeline and Meredith Newman. Smoke Alarm: Reading This may Cause Lung Cancer, Heart Disease, Emphysema, and Complicate Pregnancy . 21. Almirall, Sara and Kirsten Werne. 20 Best Spots to Smoke on Campus . 22. Werne, Kirsten. An Interview with Painted Thin . 23. Burt, Kara. All in All, We\u27re Just Paper o the Wall (Dorm Art Clue to Denison Identity) . 25. Levine, Robert. Less Talk, Moore Rock (Thurston\u27 Sound Uplifts Soul) . 30. Almirall, R.R. Turtles . 31. Almirall, RR. The Warthog Feels He Has Much in Common With Paul Newman, Others Don\u27t . 20

Cardio-metabolic risk factors and cortical thickness in a neurologically healthy male population: results from the psychological, social and biological determinants of ill health (pSoBid) study

&lt;p&gt;Introduction: Cardio-metabolic risk factors have been associated with poor physical and mental health. Epidemiological studies have shown peripheral risk markers to be associated with poor cognitive functioning in normal healthy population and in disease. The aim of the study was to explore the relationship between cardio-metabolic risk factors and cortical thickness in a neurologically healthy middle aged population-based sample.&lt;/p&gt; &lt;p&gt;Methods: T1-weighted MRI was used to create models of the cortex for calculation of regional cortical thickness in 40 adult males (average age = 50.96 years), selected from the PSOBID study. The relationship between cardio-vascular risk markers and cortical thickness across the whole brain, were examined using the general linear models. The relationship with various covariates of interest was explored.&lt;/p&gt; &lt;p&gt;Results: Lipid fractions with greater triglyceride content (TAG, VLDL and LDL) were associated with greater cortical thickness pertaining to a number of regions in the brain. Greater C reactive protein (CRP) and Intercellular adhesion molecule (ICAM-1) levels were associated with cortical thinning pertaining to perisylvian regions in the left hemisphere. Smoking status and education status were significant covariates in the model.&lt;/p&gt; &lt;p&gt;Conclusions: This exploratory study adds to a small body of existing literature increasingly showing a relationship between cardio-metabolic risk markers and regional cortical thickness involving a number of regions in the brain in a neurologically normal middle aged sample. A focused investigation of factors determining the inter-individual variations in regional cortical thickness in the adult brain could provide further clarity in our understanding of the relationship between cardio-metabolic factors and cortical structures.&lt;/p&gt