Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

BackgroundExpression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes.ResultsTen candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin.ConclusionsBestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species

Changes in chondrocyte gene expression following in vitro impaction of porcine articular cartilage in an impact injury model

Our objective was to monitor chondrocyte gene expression at 0, 3, 7, and 14 days following in vitro impaction to the articular surface of porcine patellae. Patellar facets were either axially impacted with a cylindrical impactor (25 mm/sec loading rate) to a load level of 2000 N or not impacted to serve as controls. After being placed in organ culture for 0, 3, 7, or 14 days, total RNA was isolated from full thickness cartilage slices and gene expression measured for 17 genes by quantitative real-time RT-PCR. Targeted genes included those encoding proteins involved with biological stress, inflammation, or anabolism and catabolism of cartilage extracellular matrix. Some gene expression changes were detected on the day of impaction, but most significant changes occurred at 14 days in culture. At 14 days in culture, 10 of the 17 genes were differentially expressed with col1a1 most significantly up-regulated in the impacted samples, suggesting impacted chondrocytes may have reverted to a fibroblast-like phenotype

High Diet Quality Is Associated with a Lower Risk of Cardiovascular Disease and All-Cause Mortality in Older Men

Although diet quality is implicated in cardiovascular disease (CVD) risk, few studies have investigated the relation between diet quality and the risks of CVD and mortality in older adults. This study examined the prospective associations between dietary scores and risk of CVD and all-cause mortality in older British men. A total of 3328 men (aged 60–79 y) from the British Regional Heart Study, free from CVD at baseline, were followed up for 11.3 y for CVD and mortality. Baseline food-frequency questionnaire data were used to generate 2 dietary scores: the Healthy Diet Indicator (HDI), based on WHO dietary guidelines, and the Elderly Dietary Index (EDI), based on a Mediterranean-style dietary intake, with higher scores indicating greater compliance with dietary recommendations. Cox proportional hazards regression analyses assessed associations between quartiles of HDI and EDI and risk of all-cause mortality, CVD mortality, CVD events, and coronary heart disease (CHD) events. During follow-up, 933 deaths, 327 CVD deaths, 582 CVD events, and 307 CHD events occurred. Men in the highest compared with the lowest EDI quartile had significantly lower risks of all-cause mortality (HR:0.75; 95% CI: 0.60, 0.94; P-trend = 0.03), CVD mortality (HR: 0.63; 95% CI: 0.42, 0.94; P-trend = 0.03), and CHD events(HR: 0.66; 95% CI: 0.45,0.97; P-trend = 0.05) but not CVD events (HR: 0.79; 95% CI: 0.60, 1.05;P-trend = 0.16) after adjustment for sociodemographic, behavioral, and cardiovascular risk factors. The HDI was not significantly associated with any of the outcomes. The EDI appears to be more useful than the HDI for assessing diet quality in relation to CVD and morality risk in older men. Encouraging older adults to adhere to the guidelines inherent in the EDI criteria may have public health benefit

Expensive Egos: Narcissistic Males Have Higher Cortisol

Background: Narcissism is characterized by grandiosity, low empathy, and entitlement. There has been limited research regarding the hormonal correlates of narcissism, despite the potential health implications. This study examined the role of participant narcissism and sex on basal cortisol concentrations in an undergraduate population. Methods and Findings: Participants were 106 undergraduate students (79 females, 27 males, mean age 20.1 years) from one Midwestern and one Southwestern American university. Narcissism was assessed using the Narcissistic Personality Inventory, and basal cortisol concentrations were collected from saliva samples in a laboratory setting. Regression analyses examined the effect of narcissism and sex on cortisol (log). There were no sex differences in basal cortisol, F(1,97) =.20, p =.65, and narcissism scores, F(1,97) =.00, p =.99. Stepwise linear regression models of sex and narcissism and their interaction predicting cortisol concentrations showed no main effects when including covariates, but a significant interaction, b =.27, p =.04. Narcissism was not related to cortisol in females, but significantly predicted cortisol in males. Examining the effect of unhealthy versus healthy narcissism on cortisol found that unhealthy narcissism was marginally related to cortisol in females, b =.27, p =.06, but significantly predicted higher basal cortisol in males, b =.72, p =.01, even when controlling for potential confounds. No relationship was found between sex, narcissism, or their interaction on selfreported stress

Biomechanical spinal growth modulation and progressive adolescent scoliosis – a test of the 'vicious cycle' pathogenetic hypothesis: Summary of an electronic focus group debate of the IBSE

There is no generally accepted scientific theory for the causes of adolescent idiopathic scoliosis (AIS). As part of its mission to widen understanding of scoliosis etiology, the International Federated Body on Scoliosis Etiology (IBSE) introduced the electronic focus group (EFG) as a means of increasing debate on knowledge of important topics. This has been designated as an on-line Delphi discussion. The text for this debate was written by Dr Ian A Stokes. It evaluates the hypothesis that in progressive scoliosis vertebral body wedging during adolescent growth results from asymmetric muscular loading in a "vicious cycle" (vicious cycle hypothesis of pathogenesis) by affecting vertebral body growth plates (endplate physes). A frontal plane mathematical simulation tested whether the calculated loading asymmetry created by muscles in a scoliotic spine could explain the observed rate of scoliosis increase by measuring the vertebral growth modulation by altered compression. The model deals only with vertebral (not disc) wedging. It assumes that a pre-existing scoliosis curve initiates the mechanically-modulated alteration of vertebral body growth that in turn causes worsening of the scoliosis, while everything else is anatomically and physiologically 'normal' The results provide quantitative data consistent with the vicious cycle hypothesis. Dr Stokes' biomechanical research engenders controversy. A new speculative concept is proposed of vertebral symphyseal dysplasia with implications for Dr Stokes' research and the etiology of AIS. What is not controversial is the need to test this hypothesis using additional factors in his current model and in three-dimensional quantitative models that incorporate intervertebral discs and simulate thoracic as well as lumbar scoliosis. The growth modulation process in the vertebral body can be viewed as one type of the biologic phenomenon of mechanotransduction. In certain connective tissues this involves the effects of mechanical strain on chondrocytic metabolism a possible target for novel therapeutic intervention

The Health Equity and Effectiveness of Policy Options to Reduce Dietary Salt Intake in England: Policy Forecast

Background Public health action to reduce dietary salt intake has driven substantial reductions in coronary heart disease (CHD) over the past decade, but avoidable socio-economic differentials remain. We therefore forecast how further intervention to reduce dietary salt intake might affect the overall level and inequality of CHD mortality. Methods We considered English adults, with socio-economic circumstances (SEC) stratified by quintiles of the Index of Multiple Deprivation. We used IMPACTSEC, a validated CHD policy model, to link policy implementation to salt intake, systolic blood pressure and CHD mortality. We forecast the effects of mandatory and voluntary product reformulation, nutrition labelling and social marketing (e.g., health promotion, education). To inform our forecasts, we elicited experts’ predictions on further policy implementation up to 2020. We then modelled the effects on CHD mortality up to 2025 and simultaneously assessed the socio-economic differentials of effect. Results Mandatory reformulation might prevent or postpone 4,500 (2,900–6,100) CHD deaths in total, with the effect greater by 500 (300–700) deaths or 85% in the most deprived than in the most affluent. Further voluntary reformulation was predicted to be less effective and inequality-reducing, preventing or postponing 1,500 (200–5,000) CHD deaths in total, with the effect greater by 100 (−100–600) deaths or 49% in the most deprived than in the most affluent. Further social marketing and improvements to labelling might each prevent or postpone 400–500 CHD deaths, but minimally affect inequality. Conclusions Mandatory engagement with industry to limit salt in processed-foods appears a promising and inequality-reducing option. For other policy options, our expert-driven forecast warns that future policy implementation might reach more deprived individuals less well, limiting inequality reduction. We therefore encourage planners to prioritise equity

Study on the damage mechanism of articular cartilage based on the fluid–solid coupled particle model

Articular cartilage damage is the primary cause of osteoarthritis. Very little literature work focuses on the microstructural damage mechanism of articular cartilage. In this article, a new numerical method that characterizes fluid coupling in particle model is proposed to study the damage mechanism of articular cartilage. Numerical results show that the damage mechanism of articular cartilage is related to the microstructure of collagen. The damage mechanism of superficial articular cartilage is tangential damage. On the other hand, horizontal separation is the damage mechanism of deep articular cartilage. The new numerical method shows the capability to effectively reveal the damage mechanism of superficial articular cartilage

Identification of stable normalization genes for quantitative real-time PCR in porcine articular cartilage

Abstract Background Expression levels for genes of interest must be normalized with an appropriate reference, or housekeeping gene, to make accurate comparisons of quantitative real-time PCR results. The purpose of this study was to identify the most stable housekeeping genes in porcine articular cartilage subjected to a mechanical injury from a panel of 10 candidate genes. Results Ten candidate housekeeping genes were evaluated in three different treatment groups of mechanically impacted porcine articular cartilage. The genes evaluated were: beta actin, beta-2-microglobulin, glyceraldehyde-3-phosphate dehydrogenase, hydroxymethylbilane synthase, hypoxanthine phosphoribosyl transferase, peptidylprolyl isomerase A (cyclophilin A), ribosomal protein L4, succinate dehydrogenase flavoprotein subunit A, TATA box binding protein, and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein—zeta polypeptide. The stability of the genes was measured using geNorm, BestKeeper, and NormFinder software. The four most stable genes measured via geNorm were (most to least stable) succinate dehydrogenase flavoprotein, subunit A, peptidylprolyl isomerase A, glyceraldehyde-3-phosphate dehydrogenase, beta actin; the four most stable genes measured via BestKeeper were glyceraldehyde-3-phosphate dehydrogenase, peptidylprolyl isomerase A, beta actin, succinate dehydrogenase flavoprotein, subunit A; and the four most stable genes measured via NormFinder were peptidylprolyl isomerase A, succinate dehydrogenase flavoprotein, subunit A, glyceraldehyde-3-phosphate dehydrogenase, beta actin. Conclusions BestKeeper, geNorm, and NormFinder all generated similar results for the most stable genes in porcine articular cartilage. The use of these appropriate reference genes will facilitate accurate gene expression studies of porcine articular cartilage and suggest appropriate housekeeping genes for articular cartilage studies in other species.</p

Changes in Chondrocyte Gene Expression Following In Vitro Impaction of Porcine Articular Cartilage in an Impact Injury Model

Our objective was to monitor chondrocyte gene expression at 0, 3, 7, and 14 days following in vitro impaction to the articular surface of porcine patellae. Patellar facets were either axially impacted with a cylindrical impactor (25 mm/sec loading rate) to a load level of 2000 N or not impacted to serve as controls. After being placed in organ culture for 0, 3, 7, or 14 days, total RNA was isolated from full thickness cartilage slices and gene expression measured for 17 genes by quantitative real-time RT-PCR. Targeted genes included those encoding proteins involved with biological stress, inflammation, or anabolism and catabolism of cartilage extracellular matrix. Some gene expression changes were detected on the day of impaction, but most significant changes occurred at 14 days in culture. At 14 days in culture, 10 of the 17 genes were differentially expressed with col1a1 most significantly up-regulated in the impacted samples, suggesting impacted chondrocytes may have reverted to a fibroblast-like phenotype