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

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.     

Dispersion factors in the arboreal snail Sitala jenynsi (Gastropoda: Ariophantidae)

Sitala jenynsi snails from the University and Wazo populations of Dar es Salaam, Tanzania, were released on grass, and on forest terrain, in both regions during the dry (February, March) and the rainy seasons (April). Snail dispersion was assessed as weekly quadratic means and as the overall Diffusion Coefficient 0.423 nr/week. The coefficient was enhanced by grassy habitats, continuous vegetation cover, absence of edible plants and rainy weather; conversely, dispersion was retarded by shrubs, vegetation discontinuities, presence of edible plants and dry weather. There were differences in the rate of dispersion between populations, snails from the University Campus being faster. In each release a broadly unidirectional dispersion was recorded. The populations in question, seasons and the direction of origin of the snails seemed immaterial to directionality of dispersion, The dispersion rates of Sitala jenynsi exceeded those of the helicids Cepaea nemoralis and Theba pisana and of the partulid Pariula taeniata. While observed dispersion rates of S jenynsi may warrant the mixing of individuals in a population, they seem insufficient to facilitate appreciable migration of snails between neighboring populations.     

Climate Aid Will Neither Support Forests nor the Poor: No Hope for Saving the World's Forests

Political themes undergo specific political careers. They enter and leave political agendas. Themes in development policy are, for example, biodiversity, climate change, or deforestation. While political themes change, underlying principles of aid disbursement do not. Currently the discussion on Climate Aid, in particular the reduction of emissions from deforestation and forest degradation, puts again the emphasis to deforestation. Deforestation is a prominent theme, often defined as a “forest problem,” by foreign aid policies on forestry. Foresters are once again dubbed as being able to “save the World” all on their own. In fact, it is not them who on decide the priorities of foreign (aid) policy. The decisions made about cooperating countries and thematic areas do not stem from problem pressure. Political factors dominate the program formulation and financing. Analyses of European donors' forest aid reveal that, contrary to what is defined in policies, disbursements to countries, relevant to deforestation, are not prioritized. Also the goal of poverty alleviation is not served. Most of the funds are spent in countries of already relatively high human development. Countries with “rampant” corruption are given handouts as well as small island developing countries. On the basis of these results we can draw conclusions on the ability and efficiency of (future) Climate Aid that is hoped to serve forests and the poor.     

The toxicity of Datura stramonium (thorn apple) to horses.

Meal contaminated by Datura stramonium seeds at the rate of 0.5% by weight was fed to two horses. Both horses showed clinical signs of depression, anorexia, weight loss, rapi heart and respiration rates, mydriasis, polyuria, polydipsia and diarrhoea. Both recovered with treatment. Maize screenings contaminated by the seeds had been used in the manufacture of the meal.     

The non-pathogenic Australian rabbit calicivirus RCV-A1 provides temporal and partial cross protection to lethal Rabbit Haemorrhagic Disease Virus infection which is not dependent on antibody titres

The endemic non-pathogenic Australian rabbit calicivirus RCV-A1 is known to provide some cross protection to lethal infection with the closely related Rabbit Haemorrhagic Disease Virus (RHDV). Despite its obvious negative impacts on viral biocontrol of introduced European rabbits in Australia, little is known about the extent and mechanisms of this cross protection. In this study 46 rabbits from a colony naturally infected with RCV-A1 were exposed to RHDV. Survival rates and survival times did not correlate with titres of serum antibodies specific to RCV-A1 or cross reacting to RHDV, but were instead influenced by the time between infection with the two viruses, demonstrating for the first time that the cross protection to lethal RHDV infection is transient. These findings are an important step towards a better understanding of the complex interactions of co-occurring pathogenic and non-pathogenic lagoviruses.     

Assessing the relationship between patch type and soil mites: A molecular approach

An urgent need exists for indicators of soil health and patch functionality in extensive rangelands that can be measured efficiently and at low cost. Soil mites are candidate indicators, but their identification and handling is so specialised and time-consuming that their inclusion in routine monitoring is unlikely. The aim of this study was to measure the relationship between patch type and mite assemblages using a conventional approach. An additional aim was to determine if a molecular approach traditionally used for soil microbes could be adapted for soil mites to overcome some of the bottlenecks associated with soil fauna diversity assessment. Soil mite species abundance and diversity were measured using conventional ecological methods in soil from patches with perennial grass and litter cover (PGL), and compared to soil from bare patches with annual grasses and/or litter cover (BAL). Soil mite assemblages were also assessed using a molecular method called terminal-restriction fragment length polymorphism (T-RFLP) analysis. The conventional data showed a relationship between patch type and mite assemblage. The Prostigmata and Oribatida were well represented in the PGL sites, particularly the Aphelacaridae (Oribatida). For T-RFLP analysis, the mite community was represented by a series of DNA fragment lengths that reflected mite sequence diversity. The T-RFLP data showed a distinct difference in the mite assemblage between the patch types. Where possible, T-RFLP peaks were matched to mite families using a reference 18S rDNA database, and the Aphelacaridae prevalent in the conventional samples at PGL sites were identified, as were prostigmatids and oribatids. We identified limits to the T-RFLP approach and this included an inability to distinguish some species whose DNA sequences were similar. Despite these limitations, the data still showed a clear difference between sites, and the molecular taxonomic inferences also compared well with the conventional ecological data. The results from this study indicated that the T-RFLP approach was effective in measuring mite assemblages in this system. The power of this technique lies in the fact that species diversity and abundance data can be obtained quickly because of the time taken to process hundreds of samples, from soil DNA extraction to data output on the gene analyser, can be as little as 4 days.