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

Changes in UK ophthalmology surgical training: analysis of cumulative surgical experience 2009-2015.

OBJECTIVE: To investigate changes in the patterns of cumulative surgical experience for ophthalmologists in the UK following the introduction of a new national training scheme. DESIGN: Retrospective review of all surgical training records submitted to the UK Royal College of Ophthalmologists by trainees for the award of Certificate of Completion of Training (CCT) for the period 2009-2015. SETTING: Secondary level care, UK. PARTICIPANTS: 539 trainees achieving CCT over the 7-year study period. INTERVENTIONS: Higher specialist training or ophthalmology specialist training. OUTCOME MEASURES: Number of CCT awards by years and procedures performed for cataract surgery, strabismus, corneal grafts, vitreoretinal (VR) procedures, oculoplastics and glaucoma. RESULTS: Cataract surgical experience showed little change with median number performed/performed supervised (P/PS) 592, IQR: 472-738; mean: 631. Similarly, the median number of strabismus (P/PS 34), corneal grafts (assisted, 9) and VR procedures (assisted, 34) appeared constant. There was a trend towards increasing surgical numbers for oculoplastics (median 116) and glaucoma (57). Overall case numbers for ophthalmic specialist training (OST) trainees (7-year training programme) were higher than higher surgical training (HST) trainees (4.5-year programme) with the exception of squint (P/PS), corneal grafts (P/PS) and VR cases (P/PS). CONCLUSIONS: Overall case numbers reported at time of CCT application appear stable or with a marginal trend towards increasing case numbers. HST (4.5-year programme) case numbers do not include those performed before entry to HST, and although case numbers tended to be higher for OST trainees (7-year programme) compared with HST trainees, they were not proportionately so

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

Thyroid dysfunction in preterm neonates exposed to iodine

Background: Infants <32 weeks' gestation should not be exposed to topical iodine and its avoidance is recommended during pregnancy and breast feeding. Exposure to contrast media and topical iodine is frequently used in many preterm neonates. Aim: To determine whether thyrotropin levels in preterm infants are affected by exposure to intrapartum/neonatal topical iodine and/or the use of iodinated contrast media. Design: Infants <32 weeks' gestation were recruited. Maternal and neonatal exposures to iodinated contrast media and topical iodine were recorded; levels of thyrotropin and thyroxine were measured from blood-spot cards on postnatal days 7, 14, 28 and the equivalent of 36 weeks' gestation. Results: One hundred and twenty-five infants were exposed to topical iodine/contrast media and 48 infants were unexposed. No infants were treated for hypothyroidism; three infants (exposed group) had transient hyperthyro-tropinaemia. Mean thyrotropin levels were significantly higher on postnatal days 7, 14 and 28 in infants exposed to topical iodine prior to caesarean section compared to unexposed infants, a relationship which persisted after adjustment. Conclusions: In the context of this study, neonatal thyroid dysfunction was seen following exposure to iodine via caesarean section but not via exposure to contrast medi

Experience and response to a randomised controlled trial of extended-release injectable buprenorphine versus sublingual tablet buprenorphine and oral liquid methadone for opioid use disorder:protocol for a mixed-methods evaluation

INTRODUCTION: Opioid use disorder (OUD) is a debilitating and persistent disorder. The standard-of-care treatment is daily maintenance dosing of sublingual buprenorphine (BUP-SL) or oral methadone (MET). Monthly, extended-release, subcutaneous injectable buprenorphine (BUP-XR) has been developed to enhance treatment effectiveness. This study aims to investigate the experiences of participants who have been offered BUP-XR (evaluation 1), health-related quality-of-life among participants who have opted to receive BUP-XR longer term (evaluation 2) and the experiences of participants allocated to receive BUP-XR or BUP-SL or MET with the offer of adjunctive personalised psychosocial intervention (evaluation 3). METHODS AND ANALYSIS: Three qualitative–quantitative (mixed-methods) evaluations embedded in a five-centre, head-to-head, randomised controlled trial of BUP-XR versus BUP-SL and MET in the UK. Evaluation 1 is a four-centre interview anchored on an OUD-related topic guide and conducted after the 24-week trial endpoint. Evaluation 2 is a two-centre interview anchored on medications for opioid use disorder-specific quality-of-life topic guide conducted among participants after 12–24 months. Evaluation 3: single-centre interview after the 24-week trial endpoint. All evaluations include selected trial clinical measures, with evaluation 2 incorporating additional questionnaires. Target participant recruitment for evaluations 1 and 2 is 15 participants per centre (n=60 and n=30, respectively). Recruitment for evaluation 3 is 15 participants per treatment arm (n=30). Each evaluation will be underpinned by theory, drawing on constructs from the behavioural model for health service use or the health-related quality-of-life model. Qualitative data analysis will be by iterative categorisation. ETHICS AND DISSEMINATION: Study protocol, consent materials and questionnaires were approved by the London-Brighton and Sussex research ethics committee (reference: 19/LO/0483) and the Health Research Authority (IRAS project number 255522). Participants will be provided with information sheets and informed written consent will be obtained for each evaluation. Study findings will be disseminated through peer-reviewed scientific journals. TRIAL REGISTRATION NUMBER: 2018-004460-63

Psychometric properties of leadership scales for health professionals : a systematic review

Background: The important role of leaders in the translation of health research is acknowledged in the implementation science literature. However, the accurate measurement of leadership traits and behaviours in health professionals has not been directly addressed. This review aimed to identify whether scales which measure leadership traits and behaviours have been found to be reliable and valid for use with health professionals. Methods: A systematic review was conducted. MEDLINE, EMBASE, PsycINFO, Cochrane, CINAHL, Scopus, ABI/ INFORMIT and Business Source Ultimate were searched to identify publications which reported original research testing the reliability, validity or acceptability of a leadership-related scale with health professionals. Results: Of 2814 records, a total of 39 studies met the inclusion criteria, from which 33 scales were identified as having undergone some form of psychometric testing with health professionals. The most commonly used was the Implementation Leadership Scale (n = 5) and the Multifactor Leadership Questionnaire (n = 3). Of the 33 scales, the majority of scales were validated in English speaking countries including the USA (n = 15) and Canada (n = 4), but also with some translations and use in Europe and Asia, predominantly with samples of nurses (n = 27) or allied health professionals (n = 10). Only two validation studies included physicians. Content validity and internal consistency were evident for most scales (n = 30 and 29, respectively). Only 20 of the 33 scales were found to satisfy the acceptable thresholds for good construct validity. Very limited testing occurred in relation to test-re-test reliability, responsiveness, acceptability, cross-cultural revalidation, convergent validity, discriminant validity and criterion validity. Conclusions: Seven scales may be sufficiently sound to be used with professionals, primarily with nurses. There is an absence of validation of leadership scales with regard to physicians. Given that physicians, along with nurses and allied health professionals have a leadership role in driving the implementation of evidence-based healthcare, this constitutes a clear gap in the psychometric testing of leadership scales for use in healthcare implementation research and practice

ALK1 signalling analysis identifies angiogenesis related genes and reveals disparity between TGF-β and constitutively active receptor induced gene expression

BACKGROUND: TGF-β1 is an important angiogenic factor involved in the different aspects of angiogenesis and vessel maintenance. TGF-β signalling is mediated by the TβRII/ALK5 receptor complex activating the Smad2/Smad3 pathway. In endothelial cells TGF-β utilizes a second type I receptor, ALK1, activating the Smad1/Smad5 pathway. Consequently, a perturbance of ALK1, ALK5 or TβRII activity leads to vascular defects. Mutations in ALK1 cause the vascular disorder hereditary hemorrhagic telangiectasia (HHT). METHODS: The identification of ALK1 and not ALK5 regulated genes in endothelial cells, might help to better understand the development of HHT. Therefore, the human microvascular endothelial cell line HMEC-1 was infected with a recombinant constitutively active ALK1 adenovirus, and gene expression was studied by using gene arrays and quantitative real-time PCR analysis. RESULTS: After 24 hours, 34 genes were identified to be up-regulated by ALK1 signalling. Analysing ALK1 regulated gene expression after 4 hours revealed 13 genes to be up- and 2 to be down-regulated. Several of these genes, including IL-8, ET-1, ID1, HPTPη and TEAD4 are reported to be involved in angiogenesis. Evaluation of ALK1 regulated gene expression in different human endothelial cell types was not in complete agreement. Further on, disparity between constitutively active ALK1 and TGF-β1 induced gene expression in HMEC-1 cells and primary HUVECs was observed. CONCLUSION: Gene array analysis identified 49 genes to be regulated by ALK1 signalling and at least 14 genes are reported to be involved in angiogenesis. There was substantial agreement between the gene array and quantitative real-time PCR data. The angiogenesis related genes might be potential HHT modifier genes. In addition, the results suggest endothelial cell type specific ALK1 and TGF-β signalling