BackgroundOne requisite of quantitative reverse transcription PCR (qRT-PCR) is to normalise the data with an internal reference gene that is invariant regardless of treatment, such as virus infection. Several studies have found variability in the expression of commonly used housekeeping genes, such as beta-actin (ACTB) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), under different experimental settings. However, ACTB and GAPDH remain widely used in the studies of host gene response to virus infections, including influenza viruses. To date no detailed study has been described that compares the suitability of commonly used housekeeping genes in influenza virus infections. The present study evaluated several commonly used housekeeping genes [ACTB, GAPDH, 18S ribosomal RNA (18S rRNA), ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide (ATP5B) and ATP synthase, H+ transporting, mitochondrial Fo complex, subunit C1 (subunit 9) (ATP5G1)] to identify the most stably expressed gene in human, pig, chicken and duck cells infected with a range of influenza A virus subtypes.ResultsThe relative expression stability of commonly used housekeeping genes were determined in primary human bronchial epithelial cells (HBECs), pig tracheal epithelial cells (PTECs), and chicken and duck primary lung-derived cells infected with five influenza A virus subtypes. Analysis of qRT-PCR data from virus and mock infected cells using NormFinder and BestKeeper software programmes found that 18S rRNA was the most stable gene in HBECs, PTECs and avian lung cells.ConclusionsBased on the presented data from cell culture models (HBECs, PTECs, chicken and duck lung cells) infected with a range of influenza viruses, we found that 18S rRNA is the most stable reference gene for normalising qRT-PCR data. Expression levels of the other housekeeping genes evaluated in this study (including ACTB and GPADH) were highly affected by influenza virus infection and hence are not reliable as reference genes for RNA normalisation.

Highly pathogenic avian influenza (HPAI) H5N1 viruses cause severe infection in chickens at near complete mortality, but corresponding infection in ducks is typically mild or asymptomatic. To understand the underlying molecular differences in host response, primary chicken and duck lung cells, infected with two HPAI H5N1 viruses and a low pathogenicity avian influenza (LPAI) H2N3 virus, were subjected to RNA expression profiling. Chicken cells but not duck cells showed highly elevated immune and pro-inflammatory responses following HPAI virus infection. HPAI H5N1 virus challenge studies in chickens and ducks corroborated the in vitro findings. To try to determine the underlying mechanisms, we investigated the role of signal transducer and activator of transcription-3 (STAT-3) in mediating pro-inflammatory response to HPAIV infection in chicken and duck cells. We found that STAT-3 expression was down-regulated in chickens but was up-regulated or unaffected in ducks in vitro and in vivo following H5N1 virus infection. Low basal STAT-3 expression in chicken cells was completely inhibited by H5N1 virus infection. By contrast, constitutively active STAT-3 detected in duck cells was unaffected by H5N1 virus infection. Transient constitutively-active STAT-3 transfection in chicken cells significantly reduced pro-inflammatory response to H5N1 virus infection; on the other hand, chemical inhibition of STAT-3 activation in duck cells increased pro-inflammatory gene expression following H5N1 virus infection. Collectively, we propose that elevated pro-inflammatory response in chickens is a major pathogenicity factor of HPAI H5N1 virus infection, mediated in part by the inhibition of STAT-3.Electronic supplementary materialThe online version of this article (doi:10.1186/s13567-014-0118-3) contains supplementary material, which is available to authorized users.

Point mutations in the intronic ZRS region of Lmbr1, a limb specific cis-regulatory element of Sonic hedgehog (Shh), are associated with polydactyly in humans, cats, and mice. We and others have recently mapped the dominant preaxial polydactyly (Po) locus in Silkie chickens to a single nucleotide polymorphism (SNP) in the ZRS region. Using polymorphisms in the chicken Shh sequence, we confirm that the ZRS region directly regulates Shh expression in the developing limb causing ectopic Shh expression in the anterior leg, prolonged Shh expression in the posterior limb, and allelic imbalance between wt and Slk Shh alleles in heterozygote limbs. Using Silkie legs, we have explored the consequences of increased Shh expression in the posterior leg on the patterning of the toes, and the induction of preaxial polydactyly.

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Memories are classified as consolidated (stable) or labile according to whether they withstand amnestic treatment, or not. In contrast to the general prevalence of this classification, its neuronal and molecular basis is poorly understood. Here, we focused on consolidated and labile memories induced after a single cycle training in the Drosophila aversive olfactory conditioning paradigm and we used mutants to define the impactofcAMPsignals.AtthebiochemicallevelwereportthatcAMPsignalsmisrelatedineither rutabaga(rut)ordunce (

This paper summarizes the proceedings of a workshop held at Trinity Hall, Cambridge to discuss comparability and includes additional information and references to related information added subsequently to the workshop. Comparability is the need to demonstrate equivalence of product after a process change; a recent publication states that this ‘may be difficult for cell-based medicinal products’. Therefore a well-managed change process is required which needs access to good science and regulatory advice and developers are encouraged to seek help early. The workshop shared current thinking and best practice and allowed the definition of key research questions. The intent of this report is to summarize the key issues and the consensus reached on each of these by the expert delegates.

Insects successfully occupy most environmental niches and this success depends on surviving a broad range of environmental stressors including temperature, desiccation, xenobiotic, osmotic and infection stress. Epithelial tissues play key roles as barriers between the external and internal environments and therefore maintain homeostasis and organismal tolerance to multiple stressors. As such, the crucial role of epithelia in organismal stress tolerance cannot be underestimated. At a molecular level, multiple cell-specific signalling pathways including cyclic cAMP, cyclic cGMP and calcium modulate tissue, and hence, organismal responses to stress. Thus, epithelial cell-specific signal transduction can be usefully studied to determine the molecular mechanisms of organismal stress tolerance in vivo. This review will explore cell signalling modulation of stress tolerance in insects by focusing on cell signalling in a fluid transporting epithelium -the Malpighian tubule. Manipulation of specific genes and signalling pathways in only defined tubule cell types can influence the survival outcome in response to multiple environmental stressors including desiccation, immune, salt (ionic) and oxidative stress, suggesting that studies in the genetic model Drosophila melanogaster may reveal novel pathways required for stress tolerance.