Relative transcript quantification by Quantitative PCR: Roughly right or precisely wrong?

BACKGROUND: When estimating relative transcript abundances by quantitative real-time PCR (Q-PCR) we found that the results can vary dramatically depending on the method chosen for data analysis. RESULTS: Analyses of Q-PCR results from a salmon louse starvation experiment show that, even with apparently good raw data, different analytical approaches [1,2] may lead to opposing biological conclusions. CONCLUSION: The results emphasise the importance of being cautious when analysing Q-PCR data and indicate that uncritical routine application of an analytical method will eventually result in incorrect conclusions. We do not know the extent of, or have a universal solution to this problem. However, we strongly recommend caution when analysing Q-PCR results e.g. by using two or more analytical approaches to validate conclusions. In our view a common effort should be made to standardise methods for analysis and validation of Q-PCR results

Decolonizing Higher Education: Rationales and Implementations from the Subject of Music History

In addition to ongoing territorial and material re-organization of power as a result of 19th-century European colonialism, there has been an increasing focus on decolonializing knowledge practices in higher education. Research communities are discussing what it could mean to decolonize thinking practices, conditions of knowledge creation, and access to higher education. Since the arts are a powerful tool for change, we want to contribute to the ongoing scholarly discussion by introducing examples of decolonial practices used in the Bachelor program in music performance. By presenting three cases from the subject of music history at two Norwegian universities, we provide insight into why and how we can teach differently, what kind of resistance we meet, and how we can make use of discomfort to decolonize knowledge practices. Our empirical material is composed of our own experiences, student course evaluations, conversations with students and teachers, as well as module descriptions. In the basis of our findings, we propose a strategic canonism to mediate in an increasingly polarized field

Dark neutrino interactions make gravitational waves blue

New interactions of neutrinos can stop them from free streaming in the early Universe even after the weak decoupling epoch. This results in the enhancement of the primordial gravitational wave amplitude on small scales compared to the standard $\Lambda$CDM prediction. In this paper we calculate the effect of dark matter neutrino interactions in CMB tensor $B$-modes spectrum. We show that the effect of new neutrino interactions generates a scale or $\ell$ dependent imprint in the CMB $B$-modes power spectrum at $\ell \gtrsim 100$. In the event that primordial $B$-modes are detected by future experiments, a departure from scale invariance, with a blue spectrum, may not necessarily mean failure of simple inflationary models but instead may be a sign of non-standard interactions of relativistic particles. New interactions of neutrinos also induce a phase shift in the CMB B-mode power spectrum which cannot be mimicked by simple modifications of the primordial tensor power spectrum. There is rich information hidden in the CMB $B$-modes spectrum beyond just the tensor to scalar ratio.Comment: 31 pages, 10 figures. Version published in Phys. Rev.

Transcription of reference genes used for quantitative RT-PCR in Atlantic salmon is affected by viral infection

Relative quantification using RT-qPCR is a widely used method for transcription profiling. Transcript levels of target genes in fish after experimental infection is often reported without documentation of stably transcribed reference genes. We present results demonstrating that transcription of typically used reference genes in Atlantic salmon is not stable during experimental infection with salmon pancreas disease virus (SPDV). Transcript levels 0 to 6 weeks after challenge revealed statistically significant changes between time-points that corresponded with a peak in viral load 3 weeks after challenge. The results emphasize the need for thorough method validation prior to transcriptional studies during viral infections

Salmon louse (Lepeophtheirus salmonis) transcriptomes during post molting maturation and egg production, revealed using EST-sequencing and microarray analysis

<p>Abstract</p> <p>Background</p> <p><it>Lepeophtheirus salmonis </it>is an ectoparasitic copepod feeding on skin, mucus and blood from salmonid hosts. Initial analysis of EST sequences from pre adult and adult stages of <it>L. salmonis </it>revealed a large proportion of novel transcripts. In order to link unknown transcripts to biological functions we have combined EST sequencing and microarray analysis to characterize female salmon louse transcriptomes during post molting maturation and egg production.</p> <p>Results</p> <p>EST sequence analysis shows that 43% of the ESTs have no significant hits in GenBank. Sequenced ESTs assembled into 556 contigs and 1614 singletons and whenever homologous genes were identified no clear correlation with homologous genes from any specific animal group was evident. Sequence comparison of 27 <it>L. salmonis </it>proteins with homologous proteins in humans, zebrafish, insects and crustaceans revealed an almost identical sequence identity with all species.</p> <p>Microarray analysis of maturing female adult salmon lice revealed two major transcription patterns; up-regulation during the final molting followed by down regulation and female specific up regulation during post molting growth and egg production. For a third minor group of ESTs transcription decreased during molting from pre-adult II to immature adults. Genes regulated during molting typically gave hits with cuticula proteins whilst transcripts up regulated during post molting growth were female specific, including two vitellogenins.</p> <p>Conclusion</p> <p>The copepod <it>L.salmonis </it>contains high a level of novel genes. Among analyzed <it>L.salmonis </it>proteins, sequence identities with homologous proteins in crustaceans are no higher than to homologous proteins in humans. Three distinct processes, molting, post molting growth and egg production correlate with transcriptional regulation of three groups of transcripts; two including genes related to growth, one including genes related to egg production. The function of the regulated transcripts is discussed in relation to post molting morphological changes in adult female salmon louse. There is clear evidence that transcription of the major yolk proteins is not induced before some of the post molting growth of abdomen and the genital segment has occurred. A hallmark for the observed growth is transcription of many putative cuticula proteins prior to the size increase.</p

Performance of magnetic resonance imaging-based prostate cancer risk calculators and decision strategies in two large European medical centres

Objectives: To compare the performance of currently available biopsy decision support tools incorporating magnetic resonance imaging (MRI) findings in predicting clinically significant prostate cancer (csPCa). Patients and Methods: We retrospectively included men who underwent prostate MRI and subsequent targeted and/or systematic prostate biopsies in two large European centres. Available decision support tools were identified by a PubMed search. Performance was assessed by calibration, discrimination, decision curve analysis (DCA) and numbers of biopsies avoided vs csPCa cases missed, before and after recalibration, at risk thresholds of 5%–20%. Results: A total of 940 men were included, 507 (54%) had csPCa. The median (interquartile range) age, prostate-specific antigen (PSA) level, and PSA density (PSAD) were 68 (63–72) years, 9 (7–15) ng/mL, and 0.20 (0.13–0.32) ng/mL2, respectively. In all, 18 multivariable risk calculators (MRI-RCs) and dichotomous biopsy decision strategies based on MRI findings and PSAD thresholds were assessed. The Van Leeuwen model and the Rotterdam Prostate Cancer Risk Calculator (RPCRC) had the best discriminative ability (area under the receiver operating characteristic curve 0.86) of the MRI-RCs that could be assessed in the whole cohort. DCA showed the highest clinical utility for the Van Leeuwen model, followed by the RPCRC. At the 10% threshold the Van Leeuwen model would avoid 22% of biopsies, missing 1.8% of csPCa, whilst the RPCRC would avoid 20% of biopsies, missing 2.6% of csPCas. These multivariable models outperformed all dichotomous decision strategies based only on MRI-findings and PSAD. Conclusions: Even in this high-risk cohort, biopsy decision support tools would avoid many prostate biopsies, whilst missing very few csPCa cases. The Van Leeuwen model had the highest clinical utility, followed by the RPCRC. These multivariable MRI-RCs outperformed and should be favoured over decision strategies based only on MRI and PSAD.</p

A trypsin-like protease with apparent dual function in early Lepeophtheirus salmonis (Krøyer) development

<p>Abstract</p> <p>Background</p> <p>Trypsin-like serine proteases are involved in a large number of processes including digestive degradation, regulation of developmental processes, yolk degradation and yolk degradome activation. Trypsin like peptidases considered to be involved in digestion have been characterized in <it>Lepeophtheirus salmonis</it>. During these studies a trypsin-like peptidase which differed in a number of traits were identified.</p> <p>Results</p> <p>An intronless trypsin-like serine peptidase (<it>LsTryp10</it>) from <it>L., salmonis </it>was identified and characterized. <it>LsTryp10 </it>mRNA is evenly distributed in the ovaries and oocytes, but is located along the ova periphery. LsTryp10 protein is deposited in the oocytes and all embryonic cells. <it>LsTryp10 </it>mRNA translation and concurrent degradation after fertilization was found in the embryos demonstrating that LsTryp10 protein is produced both by the embryo and maternally. The results furthermore indicate that LsTryp10 protein of maternal origin has a distribution pattern different to that of embryonic origin.</p> <p>Conclusion</p> <p>Based on present data and previous studies of peptidases in oocytes and embryos, we hypothesize that maternally deposited LsTryp10 protein is involved in regulation of the yolk degradome. The function of LsTryp10 produced by the embryonic cells remains unknown. To our knowledge a similar expression pattern has not previously been reported for any protease.</p

Effect of two constant light regimens on antibody profiles and immune gene expression in Atlantic salmon following vaccination and experimental challenge with salmonid alphavirus

Before seawater transfer, farmed Atlantic salmon are subjected to treatments that may affect the immune system and susceptibility to pathogens. E.g., exposure to constant light (CL) stimulates smoltification, which prepares salmon to life in sea water, but endocrine changes in this period are associated with suppression of immune genes. Salmon are vaccinated towards end of the freshwater period to safeguard that adequate vaccine efficacy is achieved by the time the fish is transferred to sea. In the present study, we investigated how the responses to vaccination and viral infection varied depending on the time of CL onset relative to vaccination. The salmon were either exposed to CL two weeks prior to vaccination (2-PRI) or exposed to CL at the time of vaccination (0-PRI). A cohabitant challenge with salmonid alphavirus, the causative agent of pancreatic disease, was performed 9 weeks post vaccination. The immunological effects of the different light manipulation were examined at 0- and 6-weeks post vaccination, and 6 weeks post challenge. Antibody levels in serum were measured using a serological bead-based multiplex panel as well as ELISA, and 92 immune genes in heart and spleen were measured using an integrated fluidic circuit-based qPCR array for multiple gene expression. The 2-PRI group showed a moderate transcript down-regulation of genes in the heart at the time of vaccination, which were restored 6 weeks after vaccination (WPV). Conversely, at 6WPV a down-regulation was seen for the 0-PRI fish. Moreover, the 2-PRI group had significantly higher levels of antibodies binding to three of the vaccine components at 6WPV, compared to 0-PRI. In response to SAV challenge, transcription of immune genes between 2-PRI and 0-PRI was markedly dissimilar in the heart and spleen of control fish, but no difference was found between vaccinated salmon from the two CL regimens. Thus, by using labor-saving high throughput detection methods, we demonstrated that light regimens affected antibody production and transcription of immune genes in non-vaccinated and virus challenged salmon, but the differences between the light treatment groups appeared eliminated by vaccination.publishedVersio

Phenotypic and genetic characterization of Piscirickettsia salmonis from Chilean and Canadian salmonids

Background. The study presents the phenotypic and genetic characterization of selected P. salmonis isolates from Atlantic salmon and rainbow trout suffering from SRS (salmonid rickettsial septicemia) in Chile and in Canada. The phenotypic characterization of the P. salmonis isolates were based on growth on different agar media (including a newly developed medium), different growth temperatures, antibiotics susceptibility and biochemical tests. Results. This is the first study differentiating Chilean P. salmonis isolates into two separate genetic groups. Genotyping, based on 16S rRNA-ITS and concatenated housekeeping genes grouped the selected isolates into two clades, constituted by the Chilean strains, while the Canadian isolates form a branch in the phylogenetic tree. The latter consisted of two isolates that were different in both genetic and phenotypic characteristics. The phylogenies and the MLST do not reflect the origin of the isolates with respect to host species. The isolates included were heterogeneous in phenotypic tests. Conclusions. The genotyping methods developed in this study provided a tool for separation of P. salmonis isolates into distinct clades. The SRS outbreaks in Chile are caused by minimum two different genetic groups of P. salmonis. This heterogeneity should be considered in future development of vaccines against this bacterium in Chile. Two different strains of P. salmonis, in regards to genetic and phenotypic characteristics, can occur in the same contemporary outbreak of SRS.publishedVersio

Integrating theory and practice in conservatoires: formulating holistic models for teaching and learning improvisation

Academic study has become a more significant part of a conservatoire education in recent times, but it has not always informed performance as effectively as it might. There is a need for further development of an academic curriculum that is specifically relevant to performers, in which the links between theory and practice are made explicit rather than expecting students to construct these for themselves. This article reports on research into the integration of theory and practice at Leeds College of Music, UK, using jazz improvisation as a case study. Pilot teaching sessions within two modules explored ways in which students can be encouraged to engage actively with an appropriate academic curriculum that is embedded within a holistic learning experience