An investigation into the cognitive effects of delayed visual feedback

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Expression stability of commonly used reference genes in canine articular connective tissues

<p>Abstract</p> <p>Background</p> <p>The quantification of gene expression in tissue samples requires the use of reference genes to normalise transcript numbers between different samples. Reference gene stability may vary between different tissues, and between the same tissue in different disease states. We evaluated the stability of 9 reference genes commonly used in human gene expression studies. Real-time reverse transcription PCR and a mathematical algorithm were used to establish which reference genes were most stably expressed in normal and diseased canine articular tissues and two canine cell lines stimulated with lipolysaccaride (LPS).</p> <p>Results</p> <p>The optimal reference genes for comparing gene expression data between normal and diseased infrapatella fat pad were <it>RPL13A </it>and <it>YWHAZ </it>(M = 0.56). The ideal reference genes for comparing normal and osteoarthritic (OA) cartilage were <it>RPL13A </it>and <it>SDHA </it>(M = 0.57). The best reference genes for comparing normal and ruptured canine cranial cruciate ligament were <it>B2M </it>and <it>TBP </it>(M = 0.59). The best reference genes for normalising gene expression data from normal and LPS stimulated cell lines were <it>SDHA </it>and <it>YWHAZ </it>(K6) or <it>SDHA </it>and <it>HMBS </it>(DH82), which had expression stability (M) values of 0.05 (K6) and 0.07 (DH82) respectively. The number of reference genes required to reduce pairwise variation (V) to <0.20 was 4 for cell lines, 5 for cartilage, 7 for cranial cruciate ligament and 8 for fat tissue. Reference gene stability was not related to the level of gene expression.</p> <p>Conclusion</p> <p>The reference genes demonstrating the most stable expression within each different canine articular tissue were identified, but no single reference gene was identified as having stable expression in all different tissue types. This study underlines the necessity to select reference genes on the basis of tissue and disease specific expression profile evaluation and highlights the requirement for the identification of new reference genes with greater expression stability for use in canine articular tissue gene expression studies.</p

Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data

<p>Abstract</p> <p>Background</p> <p>Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RT-qPCR) is the most accurate measure of gene expression in biological systems. The comparison of different samples requires the transformation of data through a process called normalisation. Reference or housekeeping genes are candidate genes which are selected on the basis of constitutive expression across samples, and allow the quantification of changes in gene expression. At present, no reference gene has been identified for any organism which is universally optimal for use across different tissue types or disease situations. We used microarray data to identify new reference genes generated from total RNA isolated from normal and osteoarthritic canine articular tissues (bone, ligament, cartilage, synovium and fat). RT-qPCR assays were designed and applied to each different articular tissue. Reference gene expression stability and ranking was compared using three different mathematical algorithms.</p> <p>Results</p> <p>Twelve new potential reference genes were identified from microarray data. One gene (mitochondrial ribosomal protein S7 [<it>MRPS7</it>]) was stably expressed in all five of the articular tissues evaluated. One gene HIRA interacting protein 5 isoform 2 [<it>HIRP5</it>]) was stably expressed in four of the tissues evaluated. A commonly used reference gene glyceraldehyde-3-phosphate dehydrogenase (<it>GAPDH</it>) was not stably expressed in any of the tissues evaluated. Most consistent agreement between rank ordering of reference genes was observed between <it>Bestkeeper©</it> and geNorm, although each method tended to agree on the identity of the most stably expressed genes and the least stably expressed genes for each tissue. New reference genes identified using microarray data normalised in a conventional manner were more stable than those identified by microarray data normalised by using a real-time RT-qPCR methodology.</p> <p>Conclusion</p> <p>Microarray data normalised by a conventional manner can be filtered using a simple stepwise procedure to identify new reference genes, some of which will demonstrate good measures of stability. Mitochondrial ribosomal protein S7 is a new reference gene worthy of investigation in other canine tissues and diseases. Different methods of reference gene stability assessment will generally agree on the most and least stably expressed genes, when co-regulation is not present.</p

Automated design analysis, assembly planning and motion study analysis using immersive virtual reality

Previous research work at Heriot-Watt University using immersive virtual reality (VR) for cable harness design showed that VR provided substantial productivity gains over traditional computer-aided design (CAD) systems. This follow-on work was aimed at understanding the degree to which aspects of this technology were contributed to these benefits and to determine if engineering design and planning processes could be analysed in detail by nonintrusively monitoring and logging engineering tasks. This involved using a CAD-equivalent VR system for cable harness routing design, harness assembly and installation planning that can be functionally evaluated using a set of creative design-tasks to measure the system and users' performance. A novel design task categorisation scheme was created and formalised which broke down the cable harness design process and associated activities. The system was also used to demonstrate the automatic generation of usable bulkhead connector, cable harness assembly and cable harness installation plans from non-intrusive user logging. Finally, the data generated from the user-logging allowed the automated activity categorisation of the user actions, automated generation of process flow diagrams and chronocyclegraphs

Expression stability of commonly used reference genes in canine articular connective tissues

Background: The quantification of gene expression in tissue samples requires the use of reference genes to normalise transcript numbers between different samples. Reference gene stability may vary between different tissues, and between the same tissue in different disease states. We evaluated the stability of 9 reference genes commonly used in human gene expression studies. Realtime reverse transcription PCR and a mathematical algorithm were used to establish which reference genes were most stably expressed in normal and diseased canine articular tissues and two canine cell lines stimulated with lipolysaccaride (LPS). Results: The optimal reference genes for comparing gene expression data between normal and diseased infrapatella fat pad were RPL13A and YWHAZ (M = 0.56). The ideal reference genes for comparing normal and osteoarthritic (OA) cartilage were RPL13A and SDHA (M = 0.57). The best reference genes for comparing normal and ruptured canine cranial cruciate ligament were B2M and TBP (M = 0.59). The best reference genes for normalising gene expression data from normal and LPS stimulated cell lines were SDHA and YWHAZ (K6) or SDHA and HMBS (DH82), which had expression stability (M) values of 0.05 (K6) and 0.07 (DH82) respectively. The number of reference genes required to reduce pairwise variation (V) to <0.20 was 4 for cell lines, 5 for cartilage, 7 for cranial cruciate ligament and 8 for fat tissue. Reference gene stability was not related to the level of gene expression. Conclusion: The reference genes demonstrating the most stable expression within each different canine articular tissue were identified, but no single reference gene was identified as having stable expression in all different tissue types. This study underlines the necessity to select reference genes on the basis of tissue and disease specific expression profile evaluation and highlights the requirement for the identification of new reference genes with greater expression stability for use in canine articular tissue gene expression studies

Analysis of normal and osteoarthritic canine cartilage mRNA expression by quantitative polymerase chain reaction

The molecular basis to mammalian osteoarthritis (OA) is unknown. We hypothesised that the expression of selected proteases, matrix molecules, and collagens believed to have a role in the pathogenesis of OA would be changed in naturally occurring canine OA cartilage when compared to normal articular cartilage. Quantitative (real-time) reverse transcriptase-polymerase chain reaction assays were designed measuring the expression of selected matrix molecules (collagens and small leucine-rich proteoglycans), key mediators of the proteolytic degradation of articular cartilage (metalloproteinases, cathepsins), and their inhibitors (tissue inhibitors of matrix metalloproteinases). All data were normalised using a geometric mean of three housekeeping genes, and the results subjected to power calculations and corrections for multiple hypothesis testing. We detected increases in the expression of BGN, COL1A2, COL2A1, COL3A1, COL5A1, CSPG2, CTSB, CTSD, LUM, MMP13, TIMP1, and TNC in naturally occurring canine OA. The expression of TIMP2 and TIMP4 was significantly reduced in canine OA cartilage. The patterns of gene expression change observed in naturally occurring canine OA were similar to those reported in naturally occurring human OA and experimental canine OA. We conclude that the expression profiles of matrix-associated molecules in end-stage mammalian OA may be comparable but that the precise aetiologies of OA affecting specific joints in different species are presently unknown

Atomic Spectral Features During Thermonuclear Flashes on Neutron Stars

The gravitational redshift measured by Cottam, Paerels and Mendez for the neutron star (NS) in the low-mass X-ray binary EXO 0748-676 depends on the identification of an absorption line during a type I burst as the H$\alpha$ line from hydrogenic Fe. We show that Fe is present above the photosphere as long as $\dot M>4\times 10^{-13}M_\odot {\rm yr^{-1}}$ during the burst. In this limit, the total Fe column is $N_{\rm Fe}\approx 3\times 10^{19}{\rm cm^{-2}}$ for incident material of solar abundances and only depends on the nuclear physics of the proton spallation. The Fe destruction creates many heavy elements with $Z<26$ which may imprint photo-ionization edges on the NS spectra during a radius expansion event or in a burst cooling tail. Detecting these features in concert with those from Fe would confirm a redshift measurement. We also begin to address the radiative transfer problem, and find that a concentrated Fe layer with $kT=1.2-1.4 {\rm keV}$ and column $N_{\rm Fe}= 7-20 \times 10^{20} {\rm cm}^{-2}$ (depending on the line depth) above the hotter continuum photosphere is required to create the H$\alpha$ line of the observed strength. This estimate must be refined by considerations of non-LTE effects as well as resonant line transport. Until these are carried out, we cannot say whether the Fe column from accretion and spallation is in conflict with the observations. We also show that hydrogenic Fe might remain in the photosphere due to radiative levitation from the high burst flux.Comment: Substantially revised version, to appear in Ap J Letter

A Case Control Study of Nutrient Intake Deficiencies in Patients Taking Warfarin

Introduction We previously published the case of a woman taking warfarin who was found to have scurvy, a disease caused by a deficiency of vitamin C. This led us to hypothesize that patients taking warfarin who consume a diet limited in vitamin K rich foods may be at risk for other nutrient deficiencies. To test our hypothesis, we studied dietary nutrient intake in patients taking warfarin compared to patients with heart disease not taking warfarin. Methods The warfarin (n=59) and control groups (n=24) comprised convenience samples of patients with heart disease over age 60 years. Patients completed a three-day food diary and reported use of supplements. Results Based on diet history, the most common deficiencies were vitamin D (100% both groups), vitamin E (93% warfarin, 92% control), vitamin A (71% warfarin, 71% control), vitamin K (66% warfarin, 58% control), vitamin C (58 % warfarin, 46% control) and pantothenic acid (69% warfarin, 71% control) with no significant differences in intake deficiencies between warfarin and control groups. Conclusion All of our patients had nutritional intake deficiencies. This may be due to Appalachian dietary habits and not the low vitamin K diet. It seems prudent to recommend multivitamins, however, universal multivitamin supplementation has not been supported by randomized controlled trials. More study is needed to determine the reason for poor nutritional intake in our Appalachian population and to determine whether similar results are evident in a larger sample