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

A proposed increase in retinal field-of-view may lead to spatial shifts in images

Visual information determines majority of our spatial behavior. The eye projects a 2-D image of the world on the retina. We demonstrate that when a monocular-like imaging system operates entirely with optically dense fluids, an increase in field-of-view (FOV) is observed compared to an experimental condition, where the ocular medium is optically neutral. Resulting spatial shifts in the retinal image towards the fovea complement the photoreceptor distribution pattern, incidentally revealing a new role for ocular fluids in the image space. Possible effects on the perceived egocentric object location are discussed.Comment: 13 pages, 2 figures, 1 tabl

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

Control of sedimentation by active tectonics, glaciation and contourite-depositing currents in Endurance Basin, South Georgia

Endurance Basin is an elongate broadly WNW-ESE trending basin located on the northern margin of the Scotia Sea, adjacent to the southern margin of the South Georgia micro-continent. Bathymetric and TOPAS sub-bottom profile data acquired in 2010 by the British research ship RRS James Clark Ross map this basin and its sedimentology for the first time. Endurance Basin contains a number of sub-basins and a substantial glaciogenic fan. The northern margin of Endurance Basin is formed by a series of steep slopes and intervening troughs. These are interpreted as a left-stepping en echelon array of oblique, strike-slip faults whilst the sub-basins are separated by compressional dip-slip faults. It appears that South Georgia is moving NW with respect to the basin. We interpret five seismic facies from TOPAS data, which are associated with distinct sedimentologies. The most striking units in the basin fill are: substantial contourite drifts located in the NW of the basin and on its southern margin; and two distinct mass transport deposits that pond in the centre of the basin. Combined with the known regional oceanographic setting, the contourites provide evidence of broadly eastward flowing bottom currents, entering the basin from at least two locations. Although landslide scars are present on the steep northern basin margin, the imaged mass transport deposits are interpreted to have been sourced from the glaciogenic fan, located in the SE of the basin, and from a contourite unit located on the basin’s southern margin. Sediments from these events are transported at least 40 km. The contourite drift sequence is at least 100 m thick in the west of the basin and may contain a palaeoenvironmental archive of Antarctic Cirumpolar Current (ACC) flow and the climate of South Georgia extending to the Pliocene. Such an archive would allow reconstruction of ACC flow through the Pleistocene glaciations and provide a means of linking ocean circulation and climate records in the sub-Antarctic Polar Front region

Dupuytren's: a systems biology disease

Dupuytren's disease (DD) is an ill-defined fibroproliferative disorder of the palm of the hands leading to digital contracture. DD commonly occurs in individuals of northern European extraction. Cellular components and processes associated with DD pathogenesis include altered gene and protein expression of cytokines, growth factors, adhesion molecules, and extracellular matrix components. Histology has shown increased but varying levels of particular types of collagen, myofibroblasts and myoglobin proteins in DD tissue. Free radicals and localised ischaemia have been suggested to trigger the proliferation of DD tissue. Although the existing available biological information on DD may contain potentially valuable (though largely uninterpreted) information, the precise aetiology of DD remains unknown. Systems biology combines mechanistic modelling with quantitative experimentation in studies of networks and better understanding of the interaction of multiple components in disease processes. Adopting systems biology may be the ideal approach for future research in order to improve understanding of complex diseases of multifactorial origin. In this review, we propose that DD is a disease of several networks rather than of a single gene, and show that this accounts for the experimental observations obtained to date from a variety of sources. We outline how DD may be investigated more effectively by employing a systems biology approach that considers the disease network as a whole rather than focusing on any specific single molecule

Attitudes towards the use and acceptance of eHealth technologies : a case study of older adults living with chronic pain and implications for rural healthcare

Acknowledgements The research described here is supported by the award made by the RCUK Digital Economy programme to the dot.rural Digital Economy Hub; award reference: EP/G066051/1. MC’s time writing the paper is funded by the Scottish Government’s Rural and Environmental Science and Analytical Services Division (RESAS) under Theme 8 ‘Vibrant Rural Communities’ of the Food, Land and People Programme (2011–2016). MC is also an Honorary Research Fellow at the Division of Applied Health Sciences, University of Aberdeen. The input of other members of the TOPS research team, Alastair Mort, Fiona Williams, Sophie Corbett, Phil Wilson and Paul MacNamee who contributed to be wider study and discussed preliminary findings reported here with the authors of the paper is acknowledged. We acknowledge the feedback on earlier versions of this paper provided by members of the Trans-Atlantic Rural Research Network, especially Stefanie Doebler and Carmen Hubbard. We also thank Deb Roberts for her comments.Peer reviewedPublisher PD

Kinetic Resolution in Asymmetric Epoxidation using Iminium Salt Catalysis

The first reported examples of kinetic resolution in epoxidation reactions using iminium salt catalysis are described, providing up to 99% ee in the epoxidation of racemic cis-chromenes

Methods to Obtain the Occupant Perspective

This chapter summarizes the most important methods for actively engaging occupants in the processes of designing buildings. Each stage in the building life cycle places different demands on the professional-to-occupant relationship. Both objective and subjective data are important in this relationship and raises key epistemological questions about factors that cannot be directly observed—e.g., how do we know what we know about occupant behavior? The chapter guides the reader through this intellectually dangerous terrain by suggesting that the best way to find out what people think is to ask them. Some methods discussed here are familiar to practitioners, including interviews, surveys, focus groups, and direct observation. Others are just entering widespread practice, including virtual reality simulations, ubiquitous sensors and monitoring systems, and momentary ecological assessments. Each method has strengths, weaknesses, and appropriateness for use during certain stages of the building life cycle. The key takeaways from this chapter are that (1) building designers and operators can learn much value from occupants and (2) the new skills needed to engage successfully can be quickly learned. Occupant-centric design approaches that employ these methods improve the likelihood of successful building, interface design, and occupant outcomes

How good are Global Newton methods? Part 2

Newton's method applied to certain problems with a discontinuous derivative operator is shown to be effective. A global Newton method in this setting is exhibited and its computational complexity is estimated. As an application a method is proposed to solve problems of linear inequalities (linear programming, phase 1). Using an example of the Klee-Minty type due to Blair, it was found that the simplex method (used in super-lindo) required over 2,000 iterations, while the method above required an average of 8 iterations (Newton steps) over 15 random starting values.Naval Surface Weapons Center, Dahlgren, VAhttp://archive.org/details/howgoodareglobal00goldO&MN Direct FundingApproved for public release; distribution is unlimited

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