Bone to pick: the importance of evaluating reference genes for RT-qPCR quantification of gene expression in craniosynostosis and bone-related tissues and cells

ABSTRACT: BACKGROUND: RT-qPCR is a common tool for quantification of gene expression, but its accuracy is dependent on the choice and stability (steady state expression levels) of the reference gene/s used for normalization. To date, in the bone field, there have been few studies to determine the most stable reference genes and, usually, RT-qPCR data is normalised to non-validated reference genes, most commonly GAPDH, ACTB and 18 S rRNA. Here we draw attention to the potential deleterious impact of using classical reference genes to normalise expression data for bone studies without prior validation of their stability. RESULTS: Using the geNorm and Normfinder programs, panels of mouse and human genes were assessed for their stability under three different experimental conditions: 1) disease progression of Crouzon syndrome (craniosynostosis) in a mouse model, 2) proliferative culture of cranial suture cells isolated from craniosynostosis patients and 3) osteogenesis of a mouse bone marrow stromal cell line. We demonstrate that classical reference genes are not always the most 'stable' genes and that gene 'stability' is highly dependent on experimental conditions. Selected stable genes, individually or in combination, were then used to normalise osteocalcin and alkaline phosphatase gene expression data during cranial suture fusion in the craniosynostosis mouse model and strategies compared. Strikingly, the expression trends of alkaline phosphatase and osteocalcin varied significantly when normalised to the least stable, the most stable or the three most stable genes. CONCLUSION: To minimise errors in evaluating gene expression levels, analysis of a reference panel and subsequent normalization to several stable genes is strongly recommended over normalization to a single gene. In particular, we conclude that use of single, non-validated "housekeeping" genes such as GAPDH, ACTB and 18 S rRNA, currently a widespread practice by researchers in the bone field, is likely to produce data of questionable reliability when changes are 2 fold or less, and such data should be interpreted with due caution.     

More than multiple introductions: Multiple taxa contribute to the genesis of the invasive California’s wild artichoke thistle

The history of some invasive species is so complex that their origins can be difficult to determine. One example of such invasive species is the California invasive known as “wild artichoke thistle” (Cynara cardunculus var. sylvestris), found in natural and disturbed ecosystems. Wild artichoke thistle is a Mediterranean native and the progenitor of two domesticated horticultural taxa, artichoke and cardoon. Different hypotheses regarding the origins of California plants have included introductions by 19th century Italian immigrants and the de-domestication (evolutionary reversion to wild-type morphology) of feral (escaped, free-living) cultivars. Using microsatellite markers, we compared the genetic constitutions of 12 artichoke thistle populations in California with possible progenitor populations: 17 Spanish and Italian wild populations and eight different artichoke and cardoon cultivars. Each California population was compared with its putative progenitors using STRUCTURE analysis. Our results suggest that California's artichoke thistle populations are polyphyletic. Surprisingly, two-thirds of California's populations closely matched populations from the Iberian Peninsula. Three populations matched domesticated artichoke. One population appears to have wild and cultivar hybrid ancestry. Alleles specific to Italian populations were found at low frequencies in some California plants, suggesting that Italian wild plants may have been in California, but have left a trivial genetic legacy. Given that the de-domesticated plants in this study appear to be as invasive as the wild taxon, we conclude with a discussion of the role that ferality and de-domestication may have in plant invasions.     

Constitutive expression of IL-7 receptor alpha does not support increased expansion or prevent contraction of antigen-specific CD4 or CD8 T cells following Listeria monocytogenes infection

Expression of IL-7Ralpha (CD127) has been suggested as a major determinant in the survival of memory T cell precursors. We investigated whether constitutive expression of IL-7Ralpha on T cells increased expansion and/or decreased contraction of endogenous Ag-specific CD4 and CD8 T cells following infection with Listeria monocytogenes. The results indicate that constitutive expression of IL-7Ralpha alone was not enough to impart an expansion or survival advantage to CD8 T cells responding to infection, and did not increase memory CD8 T cell numbers over those observed in wild-type controls. Constitutive expression of IL-7Ralpha did allow for slightly prolonged expansion of Ag-specific CD4 T cells; however, it did not alter the contraction phase or protect against the waning of memory T cell numbers at later times after infection. Memory CD4 and CD8 T cells generated in IL-7Ralpha transgenic mice expanded similarly to wild-type T cells after secondary infection, and immunized IL-7Ralpha transgenic mice were fully protected against lethal bacterial challenge demonstrating that constitutive expression of IL-7Ralpha does not impair, or markedly improve memory/secondary effector T cell function. These results indicate that expression of IL-7Ralpha alone does not support increased survival of effector Ag-specific CD4 or CD8 T cells into the memory phase following bacterial infection.     

A framework for scabies control

Scabies is a neglected tropical disease (NTD) that causes a significant health burden, particularly in disadvantaged communities and where there is overcrowding. There is emerging evidence that ivermectin-based mass drug administration (MDA) can reduce the prevalence of scabies in some settings, but evidence remains limited, and there are no formal guidelines to inform control efforts. An informal World Health Organization (WHO) consultation was organized to find agreement on strategies for global control. The consultation resulted in a framework for scabies control and recommendations for mapping of disease burden, delivery of interventions, and establishing monitoring and evaluation. Key operational research priorities were identified. This framework will allow countries to set control targets for scabies as part of national NTD strategic plans and develop control strategies using MDA for high-prevalence regions and outbreak situations. As further evidence and experience are collected and strategies are refined over time, formal guidelines can be developed. The control of scabies and the reduction of the health burden of scabies and associated conditions will be vital to achieving the targets set in WHO Roadmap for NTDs for 2021 to 2030 and the Sustainable Development Goals.     

Reproductive strategy of an invasive thistle: effects of adults on seedling survival

Invasive species are known for their ability to form monocultures that exclude native species, yet intraspecific interactions among invasives have not been well studied. Cynara cardunculus (L.) is an invasive perennial thistle that establishes high-density populations in coastal California grasslands. We examined the natural distribution of C. cardunculus seedlings in an established population and found that nearly 100% of seedlings grew within 2 m of adults despite an expected distribution peak at 3 m from source plants based on measured dispersal distances. We then investigated the role of mature plants in seedling survival and establishment with regard to live vegetation, litter, and seedling distance by planting seedlings at increasing distances around adults and applying removal treatments to the focal adult rosettes. We applied control (no removal), adult rosette removal (live leaves), litter removal (dead leaves), and adult rosette plus litter removal (all aboveground plant material) treatments. Seedlings experienced a higher rate of survival, measured by senescence date, and establishment, measured by return rate the following year, with all adult rosette removal treatments. Inhibition by adult rosettes was reduced with distance to 60–80 cm from the rosette, and there was little effect of adult plants between 80 and 200 cm. These results suggest that adult rosettes may inhibit conspecific seedlings at very close distances but provide a favorable environment for seedlings within nearby interspaces. This pattern may contribute to the creation and maintenance of high-density populations in C. cardunculus. Land managers seeking to control this species may improve long-term effectiveness by expanding management efforts to include a 2 m radius around adult plants and treating within 5 months of seedling emergence to prevent recruitment rather than treating adults alone.     

Natural Atypical Listeria innocua Strains with Listeria monocytogenes Pathogenicity Island 1 Genes

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.     

Influence of Methylphenidate on Eating in Obese Men

Objective: Rapid synaptic dopamine transport or reduced brain dopamine receptor signaling may influence energy intake. Methylphenidate, a dopamine reuptake inhibitor, increases brain synaptic dopamine and produces anorexia, suggesting that it may reduce energy intake. We investigated the effects of two doses of short‐acting methylphenidate on energy intake over one meal in obese adult males. Research Methods and Procedures: Nine obese males (>85th BMI percentile) ingested a placebo or a moderate dose (0.5 mg/kg) or a high dose (1.0 mg/kg) of methylphenidate in a within‐subject double‐blind acute laboratory study. One hour after ingestion, pizza consumption was measured in a naturalistic laboratory setting. Results: Participants reduced energy intake by 23% for the moderate dose vs. the placebo (p < 0.02), but there was no significant difference for the high dose vs. the moderate dose (p > 0.05). Participants consumed 34% fewer kilocalories after ingesting the lowest effective dose of methylphenidate compared with placebo (725.7 ± 404.5 vs.1095 ± 271.1 kcal, p < 0.01). Seven of nine subjects responded to the moderate dose. The increase in perceived drug effect above placebo was correlated with the reduction in energy intake for both the moderate (r = ?0.85, p = 0.004) and the high (r = ?0.75 p = 0.021) doses. Hunger scores were not different across drug doses or placebo before drug administration. Discussion: Methylphenidate reduced energy intake of a highly palatable food over one meal by one‐third in obese adult males. Dopamine transport inhibition may be an effective component of a comprehensive treatment for obesity.     

Are stacked species distribution models accurate at predicting multiple levels of diversity along a rainfall gradient?

We use observed patterns of species richness and composition of ant communities along a 1000 mm rainfall gradient in northern Australian savanna to assess the accuracy of species richness and turnover predictions derived from stacked species distribution models (S‐SDMs) and constrained by macroecological models (MEMs). We systematically sampled ants at 15 sites at 50 km intervals along the rainfall gradient in 2012 and 2013. Using the observed data, we created MEMs of species richness, composition and turnover. We built distribution models for 135 of the observed species using data from museum collections and online databases. We compared two approaches of stacking SDMs and three modelling algorithms to identify the most accurate way of predicting richness and composition. We then applied the same beta diversity metrics to compare the observed versus predicted patterns. Stacked SDMs consistently over‐predicted local species richness, and there was a mismatch between the observed pattern of richness estimated from the MEM, and the pattern predicted by S‐SDMs. The most accurate richness and turnover predictions occurred when the stacked models were rank‐ordered by their habitat suitability and constrained by the observed MEM richness predictions. In contrast with species richness, the predictions obtained by the MEM of community similarity, composition and turnover matched those predicted by the S‐SDMs. S‐SDMs regulated by MEMs may therefore be a useful tool in predicting compositional patterns despite being unreliable estimators of species richness. Our results highlight that the choice of species distribution model, the stacking method used, and underlying macroecological patterns all influence the accuracy of community assembly predictions derived from S‐SDMS.