A Panel of Stably Expressed Reference Genes for Real-Time qPCR Gene Expression Studies of Mallards (Anas platyrhynchos)

Determining which reference genes have the highest stability, and are therefore appropriate for normalising data, is a crucial step in the design of real-time quantitative PCR (qPCR) gene expression studies. This is particularly warranted in non-model and ecologically important species for which appropriate reference genes are lacking, such as the mallard—a key reservoir of many diseases with relevance for human and livestock health. Previous studies assessing gene expression changes as a consequence of infection in mallards have nearly universally used β-actin and/or GAPDH as reference genes without confirming their suitability as normalisers. The use of reference genes at random, without regard for stability of expression across treatment groups, can result in erroneous interpretation of data. Here, eleven putative reference genes for use in gene expression studies of the mallard were evaluated, across six different tissues, using a low pathogenic avian influenza A virus infection model. Tissue type influenced the selection of reference genes, whereby different genes were stable in blood, spleen, lung, gastrointestinal tract and colon. β-actin and GAPDH generally displayed low stability and are therefore inappropriate reference genes in many cases. The use of different algorithms (GeNorm and NormFinder) affected stability rankings, but for both algorithms it was possible to find a combination of two stable reference genes with which to normalise qPCR data in mallards. These results highlight the importance of validating the choice of normalising reference genes before conducting gene expression studies in ducks. The fact that nearly all previous studies of the influence of pathogen infection on mallard gene expression have used a single, non-validated reference gene is problematic. The toolkit of putative reference genes provided here offers a solid foundation for future studies of gene expression in mallards and other waterfowl.     

Genetic Evidence for a Molybdopterin-Containing Tellurate Reductase

The genetic identity and cofactor composition of the bacterial tellurate reductase are currently unknown. In this study, we examined the requirement of molybdopterin biosynthesis and molybdate transporter genes for tellurate reduction in Escherichia coli K-12. The results show that mutants deleted of the moaA, moaB, moaE, or mog gene in the molybdopterin biosynthesis pathway lost the ability to reduce tellurate. Deletion of the modB or modC gene in the molybdate transport pathway also resulted in complete loss of tellurate reduction activity. Genetic complementation by the wild-type sequences restored tellurate reduction activity in the mutant strains. These findings provide genetic evidence that tellurate reduction in E. coli involves a molybdoenzyme.     

Candidate genes associated with bud dormancy release in blackcurrant (<Emphasis Type="Italic">Ribes nigrum</Emphasis>L.)

Background  

The detrimental effects of mild winter temperatures on the consistency of cropping of blackcurrant (Ribes nigrum L.) in parts of Europe have led to increasing interest in the genetic control of dormancy release in this species. This study examined patterns of gene expression in leaf buds of blackcurrant to identify key differential changes in these profiles around the time of budbreak.     

Electrochemical Studies of Tirapazamine: Generation of the One-Electron Reduction Product

The electrochemical properties of the benzotriazine di-N-oxide, tirapazamine (SR4233), and the mono-and zero-N-oxides, SR4317 and SR4330 respectively, have been investigated in dimethylformamide and acetonitrile. The voltammetry of tirapazamine is complicated, with up to 6 reduction steps being identified, depending on the solvent. Both SR4317 and SR4330 show two reduction steps. The first reduction of all three compounds is a reversible or quasi-reversible step, which is assigned to a 1-electron addition. Cyclic voltammetric studies show that the anion radical product is stable, although the tirapazamine 1-electron addition product shows a tendency to participate in a chemical following reaction. Subsequent reduction steps are all highly irreversible in nature. The 2nd electron transfer of SR4317 results in the formation of the free base, SR4330, which is identified voltammetrically. Comparison is made with the voltammetric behaviour of quinoline and quinoline-oxide.     

Inter Subject Variability and Reproducibility of Diffusion Tensor Imaging within and between Different Imaging Sessions

The aim of these studies was to provide reference data on intersubject variability and reproducibility of diffusion tensor imaging. Healthy volunteers underwent imaging on two occasions using the same 3T Siemens Verio magnetic resonance scanner. At each session two identical diffusion tensor sequences were obtained along with standard structural imaging. Fractional anisotropy, apparent diffusion coefficient, axial and radial diffusivity maps were created and regions of interest applied in normalised space. The baseline data from all 26 volunteers were used to calculate the intersubject variability, while within session and between session reproducibility were calculated from all the available data. The reproducibility of measurements were used to calculate the overall and within session 95% prediction interval for zero change. The within and between session reproducibility data were lower than the values for intersubject variability, and were different across the brain. The regional mean (range) coefficient of variation figures for within session reproducibility were 2.1 (0.9–5.5%), 1.2 (0.4–3.9%), 1.2 (0.4–3.8%) and 1.8 (0.4–4.3%) for fractional anisotropy, apparent diffusion coefficient, axial and radial diffusivity, and were lower than between session reproducibility measurements (2.4 (1.1–5.9%), 1.9 (0.7–5.7%), 1.7 (0.7–4.7%) and 2.4 (0.9–5.8%); p<0.001). The calculated overall and within session 95% prediction intervals for zero change were similar. This study provides additional reference data concerning intersubject variability and reproducibility of diffusion tensor imaging conducted within the same imaging session and different imaging sessions. These data can be utilised in interventional studies to quantify change within a single imaging session, or to assess the significance of change in longitudinal studies of brain injury and disease.     

3D Modeling of the Lateral Ventricles and Histological Characterization of Periventricular Tissue in Humans and Mouse

The ventricular system carries and circulates cerebral spinal fluid (CSF) and facilitates clearance of solutes and toxins from the brain. The functional units of the ventricles are ciliated epithelial cells termed ependymal cells, which line the ventricles and through ciliary action are capable of generating laminar flow of CSF at the ventricle surface. This monolayer of ependymal cells also provides barrier and filtration functions that promote exchange between brain interstitial fluids (ISF) and circulating CSF. Biochemical changes in the brain are thereby reflected in the composition of the CSF and destruction of the ependyma can disrupt the delicate balance of CSF and ISF exchange. In humans there is a strong correlation between lateral ventricle expansion and aging. Age-associated ventriculomegaly can occur even in the absence of dementia or obstruction of CSF flow. The exact cause and progression of ventriculomegaly is often unknown; however, enlarged ventricles can show regional and, often, extensive loss of ependymal cell coverage with ventricle surface astrogliosis and associated periventricular edema replacing the functional ependymal cell monolayer. Using MRI scans together with postmortem human brain tissue, we describe how to prepare, image and compile 3D renderings of lateral ventricle volumes, calculate lateral ventricle volumes, and characterize periventricular tissue through immunohistochemical analysis of en face lateral ventricle wall tissue preparations. Corresponding analyses of mouse brain tissue are also presented supporting the use of mouse models as a means to evaluate changes to the lateral ventricles and periventricular tissue found in human aging and disease. Together, these protocols allow investigations into the cause and effect of ventriculomegaly and highlight techniques to study ventricular system health and its important barrier and filtration functions within the brain.     

Reproductive status and torpor of the marsupial Sminthopsis crassicaudata: Effect of photoperiod

1. 1. We investigated the effects of photoperiod on the reproductive state and the occurrence and pattern of torpor in male Sminthopsis crassicaudata.

2. 2. Testes regressed when animals were exposed to a short photoperiod (L:D 8:16) and recrudesced under a long photoperiod (L:D 16:8).

3. 3. Animals entered torpor under both photoperiods and no significant differences were observed in the frequency or physiological variables of torpor of S. crassicaudata between the short and long photoperiods.

4. 4. The differences in the response to photoperiod in thermal physiology and reproduction suggest that, unlike in many rodent species, torpor and reproduction in S. crassicaudata are controlled by separate environmental cues and mechanisms.

     

Connectivity and Dispersal Patterns of Protected Biogenic Reefs: Implications for the Conservation of Modiolus modiolus (L.) in the Irish Sea

Biogenic reefs created by Modiolus modiolus (Linnaeus, 1758) (horse mussel reefs) are marine habitats which support high levels of species biodiversity and provide valuable ecosystem services. Currently, M. modiolus reefs are listed as a threatened and/or declining species and habitat in all OSPAR regions and thus are highlighted as a conservation priority under the EU Marine Strategy Framework Directive (MSFD). Determining patterns of larval dispersal and genetic connectivity of remaining horse mussel populations can inform management efforts and is a critical component of effective marine spatial planning (MSP). Larval dispersal patterns and genetic structure were determined for several M. modiolus bed populations in the Irish Sea including those in Wales (North Pen Llŷn), Isle of Man (Point of Ayre) and Northern Ireland (Ards Peninsula and Strangford Lough). Simulations of larval dispersal suggested extant connectivity between populations within the Irish Sea. Results from the genetic analysis carried out using newly developed microsatellite DNA markers were consistent with those of the biophysical model. Results indicated moderately significant differentiation between the Northern Ireland populations and those in the Isle of Man and Wales. Simulations of larval dispersal over a 30 day pelagic larval duration (PLD) suggest that connectivity over a spatial scale of 150km is possible between some source and sink populations. However, it appears unlikely that larvae from Northern Ireland will connect directly with sites on the Llŷn or Isle of Man. It also appears unlikely that larvae from the Llŷn connect directly to any of the other sites. Taken together the data establishes a baseline for underpinning management and conservation of these important and threatened marine habitats in the southern part of the known range.